6,7-Oxygenated steroids and uses related thereto

ABSTRACT

Steroid compounds having various oxygen substitution on the steroid nucleus are disclosed. A specific functionality present on many of the steroid compounds is oxygen substitution at both of positions 6 and 7. Thus, certain steroids have oxygen substitution at C6 and C7, and some have specific stereochemistries such as 6α and 7β oxygen substitution, and an alpha hydrogen at the 5 position in addition to having 6α and 7β oxygen substitution. Steroids having 3,4-epoxy functionality are also disclosed. In addition, steroids having C17 pyran and δ-lactone functionality, with oxygen substitution at C6 and C7, or at C15, of the steroid nucleus, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/471,827 filed Dec. 23, 1999, (now U.S. Pat. No. 6,706,701); which isa divisional of U.S. patent application Ser. No. 08/893,575 filed Jul.10, 1997 (now U.S. Pat. No. 6,046,185); which is a continuation of U.S.patent application Ser. No. 08/679,642 filed Jul. 12. 1996 (nowabandoned); which claims the benefit of U.S. Provisional PatentApplication No. 60/023,450 filed Jul. 11, 1996, which applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The invention is directed to steroid compounds, and in particular to6,7-oxygenated steroid compounds and therapeutic uses related thereto.

BACKGROUND OF THE INVENTION

Asthma and allergy are closely related with good evidence from clinicalstudies demonstrating a strong correlation between the severity ofasthma and the degree of atopy (allergy). Sensitization to allergens isbelieved to be the most important risk factor for asthma in bothchildren and adults with approximately 90% of asthma cases exhibitingatopy.

Allergy is characterized by an increased blood serum IgE (antibody)level. Repeated exposure to allergens, in a process calledsensitization, is normally required to elicit sufficient B cellproduction of IgE specific to a given allergen or series of allergens totrigger atopy and the subsequent asthmatic or allergic response. Once Bcells are exposed to allergens. they produce antibodies which bind tothe surface of mast cells. The crosslinking of 2 antibodies by theantigen causes a series of reactions causing degranulation and therelease of a number of mediators which modulate the inflammatoryresponse. Mediators that are released or generated during the asthmaticand allergic response include histamine, leukotrienes, prostaglandins,cytokines and tryptase.

Asthma is characterized by hyperresponsiveness of the airways, episodicperiods of bronchospasm and chronic inflammation of the lungs.Obstruction of the airways is reversible with time or in response todrug therapies. Patients exhibiting normal airflow may be hyperreactiveto a variety of naturally occurring stimuli, e.g., cold air, exercise,chemicals and allergen. The most common event initiating an asthmaticresponse is an immediate hypersensitivity to common allergens includingragweed pollen, grass pollen, various fungi, dust mites, cockroaches anddomestic animals. The symptoms of the disease include chest tightness,wheezing, shortness of breath and coughing. Mild forms of the diseaseoccur in up to 10% of the U.S. population, while the U.K., Australia andNew Zealand report higher prevalences. Asthma incidence and mortalityhas been increasing worldwide, doubling over the past 20 years despitemodern therapies.

The response of the airways to allergen is complex and consists of anearly asthmatic response (EAR) which peaks 20–30 min after exposure tothe stimuli, is characterized by bronchoconstriction and normallyresolves after 1½ to 2 hours. The late asthmatic response (LAR)generally occurs 3–8 hours after initial exposure, and involves bothbronchoconstriction and the development of inflammation and edema in thelung tissue. This inflammation often becomes chronic, with epithelialdamage occurring and infiltration of the lungs with inflammatory cellssuch as eosinophils and neutrophils.

Current Treatments for Asthma

Glucocorticosteroids (steroids) are the most effective long-term therapyfor the treatment of asthma. Oral steroids are not very useful for thecontrol of acute asthma attacks and their chronic use in the control ofasthma is minimal due to the introduction of inhaled steroids. Due tothe presence of airway inflammation even in mild asthma, inhaledsteroids are used even in early stage drug therapy. As effective asinhaled steroids are, side effects limit their use and combinationtherapy is often employed. Combination therapy is divided into thefollowing areas: anti-inflammatory drugs (e.g., inhaled and oralsteroids), bronchodilators, (e.g., β₂-agonists, xanthines,anticholinergics), and mediator inhibitors (e.g., cromolyns andleukotriene antagonists).

Cromolyns (e.g., disodium cromoglycate and nedocromil) inhibit therelease of histamine in vitro and prevent bronchial hyperreactivity,while displaying few side effects. They are not effective orally andhave no bronchodilator effect. Usually chronic treatment (several days)is required to achieve optimal anti-inflammatory effect, thoughcromolyns exhibit beneficial effects against exercise-induced asthmawhen administered only 10 minutes prior to exercise. Cromolyns are, atbest, only marginally effective against moderate to severe asthma.

Glucocorticosteroids (steroids) have profound effects against lunginflammation, and are by far the most effective drugs for the treatmentof asthma and allergies. In mast cells they inhibit the production ofarachidonic acid metabolites (leukotrienes and prostaglandins) andcytokines. Responses to inhaled steroids or systemic steroids can occurwithin 4 hours but may take several days depending on the severity ofthe disease state. Symptoms often return without regular chronictreatment. Side effects of inhaled steroids used on a continual basisinclude dysphonia, local irritation and oral candidiasis (a fungalinfection). Higher doses of inhaled steroids cause suppression of theHPA-axis which is responsible for the regulation of serum cortisollevels, metabolism, stress, CNS function and immunity. Continuous use ofhigh dose inhaled steroids or oral steroids induce more severe sideeffects: severe suppression of the HPA axis, causing effects on theimmune system, hypertension, osteoporosis, peptic ulcers, growthretardation in children, behavioral problems, reproductive problems,cataracts and hematological disorders.

Beta-agonists reverse the bronchospasm produced during an asthmaticattack and have a modest activity against the onset of the response.Their routes of administration and duration of action are variable.Prolonged use of these agents can cause decreased response to thetherapy itself with the development of tolerance. These compounds haveno effect on the inflammatory response itself.

Xanthines, which are cyclic AMP phosphodiesterase inhibitors, are alsoused in bronchodilator therapy. Though effective, xanthine activity isinfluenced by a number of factors including food, age, smoking, etc. Thetherapeutic window is relatively narrow and side effects includegastrointestinal disorders, CNS disturbances, headache, anxiety andcardiac arrhythmias. The importance of treatment of inflammation inasthma and allergy has led to a decline in the use of xanthines fortherapy.

Anticholinergic agents such as ipratropium bromide are used to block thecontraction of bronchial smooth muscle induced by acetylcholine releasedas a neurotransmitter. Some positive effects are reported in asthma,with these drugs being most effective against chronic obstructivepulmonary disease. A large number of side effects are seen with thesedrugs including urinary retention, dry mouth, tachycardia, nausea,vomiting, flushing and hypertension.

Inhibitors of 5-lipoxygenase inhibit the generation of leukotrienes,while leukotriene antagonists prevent the action of leukotrienes, whichare potent bronchospastic mediators released during an asthmaticreaction. Use of leukotriene synthesis inhibitors has been associatedwith increased liver enzymes, indicating the need to monitor liverfunction closely in certain patient populations. Leukotriene inhibitorshave shown comparative activity to the cromolyns, and activityequivalent to low dose corticosteroids.

In general, moderate to severe asthma patients are poorly served by thepresent armamentarium of drugs. Drugs that are safe are only marginallyeffective, while effective drugs have unacceptable side effects withextensive monitoring of patients required. There is a significant needfor therapeutic agents that achieve safe and effective relief of asthmaand allergy symptoms. The present invention provides these and relatedbenefits as described herein.

SUMMARY OF THE INVENTION

One aspect of the invention provides compounds of the formula:

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

C17 is substituted according to any of (c), (d), (e), (f), (g), (h) and(i):

(c) ═C(R²)(R³except when C14 is substituted with methyl;

(d) —R⁵ and —OR⁶ so long as the A and B rings are not aromatic, and whenC10 is substituted with methyl then C5 is not bonded directly to oxygen,where R⁵ and R⁶ may together form a direct bond so C17 is a carbonylgroup, or may together with C17 form a cyclic 3–6 membered ether or 4–6membered lactone; otherwise R⁵ is R⁴ or —OR⁶ and R⁶ is R¹ or R⁴.

(e) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6, as long as oneof the following conditions i), ii), iii) or iv) apply:

-   -   i) C5 is substituted with a hydrogen in the alpha configuration,        and C3 is not bonded to oxygen, and when C3 is substituted with        two hydrogen atoms then C17 is not substituted with either        —CH(CH₃)(CH₂)₃CH(CH₃)₂ or —CH(CH₃)(CH₂)2C(═O)OCH₃;    -   ii) C10 and C13 are not simultaneously substituted with methyl,        and when C10 is substituted with methyl, then C14 is not        substituted with a methyl, and the A ring is never aromatic;    -   iii) if C3 and C4 are bonded to oxygen atoms, and the C6 —OR¹        substituent has the alpha configuration, and the C7 —OR¹        substituent has the beta configuration, then C17 is not        substituted with any of the following:

-   -   iv) C3 and C4 are each bonded to the same oxygen atom so as to        form an oxirane ring, with the proviso that C7 does not have        carbonyl substitution when C5 has hydroxyl or —OR¹ substitution;

(f) two of the following substituents, which are independently selected:—X, —R⁴ and —OR¹, as long as one of the above conditions i), ii), iii)or iv) apply;

(g) a cyclic structure of the formula

wherein G is —C(═O)—, —CH(OR¹)—, —C(R⁴)(OR¹)— or —C(OR¹)(OR¹)—, as longas C3 and C4 are not simultaneously substituted with hydroxyl orprotected hydroxyl;

(h) two hydrogen atoms, as long as C3 is not substituted with a carbonylgroup;

(i) one hydrogen atom and one group selected from C₁–C₃₀ hydrocarbylgroups and C₁–C₃₀ halogen substituted hydrocarbyl groups, excluding—CH(CH₃)(CH₂)₃CH(CH₃)₂;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R², R³ and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In a preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10 and C13 is independently substituted with one of—X, —R⁴ or —OR¹;

C14 is substituted with —X, —OR¹, or —R⁴ excluding methyl;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R², R³ and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof;wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of—X, —R⁴ or —OR¹;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated, with the proviso that neither the A norB ring is aromatic;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

R⁵ and R⁶ may together form a direct bond so C17 is a carbonyl group, ormay together with C17 form a cyclic 3–6 membered ether or 4–6 memberedlactone; otherwise R⁵ is R⁴ or —OR⁶ and R⁶ is R¹ or R⁴; and

X represents fluoride, chloride, bromide and iodide.

with the proviso that when C10 is substituted with methyl, then C5 isnot directly bonded to an oxygen atom.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C8, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

C3 is substituted with one of ═C(R⁴)(R⁴) and —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)—wherein n ranges from 1 to about 6, or two of —X, and —R⁴ with theproviso that C3 is not bonded to an oxygen atom, and when C3 issubstituted with two hydrogen atoms then C17 is not substituted witheither —CH(CH₃)(CH₂)₃CH(CH₃)₂ or —CH(CH₃)(CH₂)2C(═O)OCH₃;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represent acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur; where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

with the provisos that (a) C10 and C13 are not simultaneouslysubstituted with methyl, and (b) when C10 is substituted with methyl,then C14 is not substituted with a methyl;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated with the proviso that the A ring is notaromatic;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represent acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur; where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C11, C12, C15, C16 and C17 is independently substitutedwith

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

with the proviso that C17 is not substituted with any of the following:

each of C5, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

C8 is substituted with —X or —R⁴ and is preferably not bonded directlyto oxygen;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur; where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

with the proviso that C3 and C4 are not simultaneously substituted withhydroxyl or protected hydroxyl, and are preferably not simultaneouslysubstituted with oxygen atoms;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

G is —C(═O)—, —CH(OR¹)—, —C(R⁴)(OR¹)— or —C(OR¹)(OR¹)—;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another preferred embodiment, the compounds have the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C11, C12, C15, C16 and C17 is independently substitutedwith

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide;

with the proviso that C7 does not have carbonyl substitution when C5 hashydroxyl or —OR¹ substitution.

In another preferred embodiment, the compounds have one of the formulas

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12 and C16 is independently substitutedaccording to (a) or (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6,

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

C5 is substituted with a hydrogen atom;

each of C6, C7, C8, C9, C10, C13 and C14 is independently substitutedwith one of —X, —R⁴ or —OR¹; and

C17 is substituted according to (c), (d), (e) or (f):

(c) two substituents selected from hydrogen, halogen, C₁–C₃₀ saturatedhydrocarbyl excluding —CH(CH₃)(CH₂)₃CH(CH₃)₂, halogen substituted C₁–C₃₀saturated hydrocarbyl, C₁–C₃₀ unsaturated hydrocarbyl, and halogensubstituted C₁–C₃₀ unsaturated hydrocarbyl;

(d) one substituent selected from ═C(R⁴)(R⁴) with the proviso that C14is not substituted with methyl;

(e) at least one oxygen atom-containing substituent selected from ═O,—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6, —OH, and —OR¹;

(f) at least one nitrogen atom-containing substituent selected from—N(R⁴)(R⁴) wherein the two R⁴ groups may together with the nitrogen atomform one or more rings, so that the nitrogen atom-containing substituentincludes nitrogen atom-containing heterocyclic groups; wherein

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where —OR¹ groups bonded to adjacent carbon atoms may togetherform a cyclic structure which protects both hydroxyl groups;

R⁴ at each occurrence is independently selected from H and R⁵;

R⁵ is a C₁₋₃₀ organic moiety that may optionally contain at least oneheteroatom selected from the group consisting of boron, halogen,nitrogen, oxygen, silicon and sulfur; where two geminal R⁵ groups maytogether form a ring with the carbon atom to which they are both bonded;and

X represents fluoride, chloride, bromide or iodide.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound according any of the descriptions provided above,in combination with a pharmaceutically acceptable carrier or diluent.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound in combination with a pharmaceutically acceptablecarrier or diluent, the compound having the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with —X, —R⁴ and —OR¹;

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with a substituent selected from (a) or (b), wherein

(a) represents one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(T⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)—, wherein n ranges from 1 to about 6; and

(b) represents two of: —X, —R⁴ and —OR¹, which are independentlyselected at each occurrence;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where the C6 and C7 —OR¹ groups may together form a cyclicstructure which protects both hydroxyl groups;

R⁴ at each occurrence is independently selected from H and R⁵;

R⁵ is a C₁₋₃₀ organic moiety that may optionally contain at least oneheteroatom selected from the group consisting of boron, halogen,nitrogen, oxygen, silicon and sulfur; where two geminal R⁴ groups maytogether form a ring with the carbon atom to which they are both bonded;and

X represents fluoride, chloride, bromide or iodide;

with the proviso that C15 is not bonded to an oxygen atom.

In another aspect, the invention provides for the use of the abovecompounds (any one or mixture thereof) for manufacture of a medicamentfor the treatment of asthma, allergy, inflammation including arthritis,and/or thrombosis, or for treating a condition associated with anelevated level of NFκB.

In another aspect, the invention provides a process for treating asthmacomprising administering to a subject in need thereof an effectiveamount of the compound or salt thereof, or a pharmaceutical composition,each as described above.

In another aspect, the invention provides a process for treating allergycomprising administering to a subject in need thereof an effectiveamount of the compound or salt thereof, or a pharmaceutical composition,each as described above.

In another aspect, the invention provides a process for treatinginflammation due to arthritis comprising administering to a subject inneed thereof an effective amount of the compound or salt thereof, or apharmaceutical composition, each as described above.

In another aspect, the invention provides a process for treatingthrombosis comprising administering to a subject in need thereof aneffective amount of the compound or salt thereof, or a pharmaceuticalcomposition, each as described above.

In another aspect, the invention provides a process for treating acondition associated with an elevated level of NFκB activity in asubject, comprising administering to a subject in need thereof aneffective amount of the compound or salt thereof, or a pharmaceuticalcomposition, each as described above.

In another aspect, the invention provides a process for introducing anexocyclic olefin group to the C17 position of a 6,7-dioxygenated steroidcomprising providing a compound of Formula (10), reacting the compoundof Formula (10) with a Wittig reagent of Formula (11) in the presence ofa base, to provide an olefin compound of Formula (12)

wherein each of the compounds of Formulas (10) and (12) includepharmaceutically acceptable salts and solvates thereof, and wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

Ra, Rb and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide, which isindependently selected at each occurrence.

In another aspect, the invention provides a process for introducing6α,7β-dioxygenation into a steroid, comprising providing a steroid ofFormula (13) having a carbonyl group at C7 and a double bond between C5and C6, comprising a reduction the carbonyl group to a hydroxyl group,followed by a hydroboration of the double bond to provide a hydroxylgroup at C6, wherein the C6 hydroxyl group has the α-configuration andthe C7 hydroxyl group has the β-configuration,

wherein each of the compounds of Formulas (13) and (14) includepharmaceutically acceptable salts and solvates thereof, and wherein:

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C8, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In another aspect, the invention provides a process for astereocontrolled introduction of a hydroxyl group at C3 of a steroidnucleus, comprising providing a steroid compound of Formula (15) havinga carbonyl group at C3, and reducing the carbonyl group to a hydroxylgroup with a reducing agent so as to provide at least one compound ofFormulas (16) and (17)

wherein each of the compounds of Formulas (15), (16) and (17) includepharmaceutically acceptable salts and solvates thereof, and wherein:

each of C1, C2, C4, C11, C12, C15, C16 and C17 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to various steroid derivatives having specificfunctionality as described in detail herein. The compounds describedherein demonstrate effectiveness as good controllers of the asthmaticand allergic responses in that they show efficacy against mast celldegranulation, inhibition of allergen-induced bronchospasm (acute phase)and inhibition of allergen-induced lung inflammation (late phase). Thisgroup of compounds represents a new series of agents which havepotential therapeutic benefit in the treatment of asthma and allergies,with high potency, a broad spectrum of activity and the reducedprobability of side effects.

For convenience in identifying the novel features of the inventedcompounds, an unsubstituted steroid nucleus having each ring carbonthereof identified with a unique number is shown below as Structure 1.This numbering system will be used consistently herein.

The compounds of the present invention contain at least two asymmetriccarbon atoms and thus exist as enantiomers and diastereomers. Unlessotherwise noted, the present invention includes all enantiomeric anddiastereomeric forms of the compounds of the above formula. Purestereoisomers, mixtures of enantiomers and/or diastereomers, andmixtures of different compounds of the above formulae are includedwithin the present invention.

The synthesis procedures described herein, especially when taken withthe general knowledge in the art, provide sufficient guidance to thoseof ordinary skill in the art to perform the synthesis, isolation, andpurification of the preferred compounds described herein and otheranalogous compounds. Individual enantiomers may be obtained, if desired,from mixtures of the different forms by known methods of resolution,such as the formation of diastereomers, followed by recrystallization.

The compounds of the above formula may be in the form of a solvate or apharmaceutically acceptable salt, e.g., an acid addition salt. Suchsalts include hydrochloride, sulfate, phosphate, citrate, fumarate,methanesulfonate, acetate, tartrate, maleate, lactate, mandelate,salicylate, succinate and other salts known in the art.

A compound of the present invention may be prepared as a composition bycombining it with a pharmaceutically acceptable carrier or diluent.Suitable carriers or diluents include physiological saline. It will beevident to those of ordinary skill in the art that a composition of thepresent invention may contain more than one steroid compound or one ormore steroid compounds in combination with one or more non-steroidcompounds.

A specific functionality present on many of the steroid compounds of theinvention is oxygen substitution at both of positions 6 and 7. Thus,certain steroids of the invention have the oxygen substitution patternshown in Structure 2 below. Some of these steroids are additionallycharacterized by having specific stereochemistries. For example,steroids having 6α and 7β oxygen substitution, as shown in Structure 3,and steroids having an alpha hydrogen at the 5 position in addition tohaving 6α and 7β oxygen substitution, as shown in Structure 4 below,fall within the scope of the invention.

In Structures 2, 3 and 4, each of the oxygen atoms that are bonded tocarbons 6 and 7 are simultaneously bonded to an “R¹” group. The R¹ groupis hydrogen or a protecting group for an hydroxyl group. Suitableprotecting groups are set forth in Greene, “Protective Groups in OrganicChemistry”, John Wiley & Sons, New York N.Y. (1981). When a compound ofStructures 2–4 contains vicinal —OR¹ groups (i.e., —OR¹ groups onneighboring carbon atoms), those vicinal —OR¹ groups may together form acyclic structure which protects vicinal hydroxyl groups. A ketal is anexample of protected vicinal —OR¹ group. Geminal —OR¹ groups (i.e., two—OR¹ groups on the same carbon atom) may together form a cyclicstructure which protects a carbonyl group. A ketal is an example of sucha cyclic structure. It should be understood that either or both of —OR¹at C6 and C7 represents a carbonyl or protected carbonyl group, and thusat C6 and C7, R1 may be a direct bond between the oxygen atom and thecarbon (C6 or C7) to which the oxygen atom is bonded.

Steroids of the invention may have substituents with either the α or βstereochemistry at the C8 and/or C9 positions. A hydrogen atom at C8 ofthe steroids of the invention is typically in the β configuration. Inaddition, preferred steroids of the invention may have methylsubstituents with β stereochemistry at the C10 and/or C13 positions.Compounds of the invention preferably have a C14 hydrogen with the αstereochemistry when C15 is not a ketone. In preferred steroids of theinvention that have a substituent at C17, the C17 substituent has βstereochemistry.

Steroids having 6,7-dioxygenation in the B-ring according to Structure 2can be synthesized from a number of commercially available steroidalprecursors having an α,β-unsaturated carbonyl group in the A-ring,including 4-androsten-3,17-dione (compound 1 below) anddehydroisoandrosterone (compound 247 below). These specific steroidprecursors are available from Steraloids Inc., Wilton, N. H. Othersuitable steroid precursors having C3 oxygen functionalities and Δ⁵carbon-carbon double bonds may be obtained from, e.g., Aldrich ChemicalCo., Milwaukee, Wis.

An exemplary synthetic sequence to prepare a compound of Structure 2from 4-androsten-3,17-dione is summarized in Scheme 1 below.

Initially, the carbonyl functionalities of 4-androsten-3,17-dione areprotected by carbonyl protecting groups. As shown in Scheme 1, this maybe accomplished by reacting compound 1 with a benzene solution of(CH₂OH)₂ and p-TsOH, thereby converting the carbonyl groups to ketalgroups. Other suitable carbonyl protecting groups are listed in Greene,“Protective Groups in Organic Chemistry”, John Wiley & Sons, New York,N.Y. (1981). Under the acidic conditions which form the protected ketonegroups, there occurs concomitant migration of the C4–C5 carbon-carbondouble bond to the C5–C6 position, to ultimately form compound 2.

Allylic oxidation of the C5–C6 carbon-carbon double bond of compound 2introduces a carbonyl oxygen at C7, to thereby form compound 3. A numberof oxidizing agents and experimental conditions can be used for thisallylic oxidation, including chromium trioxide/3,5-dimethylpyrazolecomplex, pyridinium chlorochromate (PCC), pyridinium dichromate (PDC),or RuCl₃ and t-butylhydroperoxide.

Reduction of the resultant C7 ketone with an appropriate reducing agentgives the hydroxyl functionality at C7, as shown in compound 4. Any ofseveral metal hydride reducing agents can be used for this taskincluding sodium borohydride or lithium aluminum hydride. Generally,reduction of the C7 ketone produces the β—OH configuration by hydrideattack from the least hindered face of the steroid. The C7 hydroxylgroup is then preferably protected with an hydroxyl protecting group,e.g., t-butyldimethylsilane (TBDMS), to provide a protected allylicalcohol as in compound 5. Other suitable hydroxyl protecting groups arelisted in Greene, supra.

Introduction of the C6 oxygen can be achieved, before or afterprotection of the C7 hydroxyl group, by methods such ashydroboration/oxidation or epoxidation followed by ring opening. Forexample, the Δ⁵ carbon-carbon double bond of compound 5 can beepoxidized with any of a number of peracids including m-chloroperbenzoicacid, trifluoroperacetic acid or 3,5-dinitroperoxybenzoic acid, toprovide an epoxide such as in compound 6. Generally, the epoxideintroduced has the α-configuration arising from attack on the leasthindered face of the steroid ring structure. Subsequent ring opening ofthe epoxide can be accomplished under acidic conditions, such as 80%aqueous acetic acid at 60° C. The crude mixture contains both compound 7(having an allylic alcohol at the C6 position with the α-configuration)and the C7 silyl derivative thereof. This crude mixture can be treatedwith tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) to givea single compound (7). Alternatively, hydroboration of the Δ⁵ doublebond with an appropriate borane complex followed by oxidation usingreagents such as basic hydrogen peroxide will also introduce an hydroxylgroup in the α-configuration at C6.

Compound 7 is exemplary of compounds having the oxygenation pattern ofStructures 2 and 3. The methodology by which compound 1 may be convertedto a compound of Structures 2 and/or 3 is generally applicable to a widevariety of compounds having an α,β-unsaturated carbonyl group in theA-ring of a steroid. Additional compounds of Structures 2 and/or 3 maybe prepared by modification of a dihydroxy compound such as compound 7.In such case, it may be necessary to protect each of the C6 and C7hydroxyl groups, and methodology to achieve such protection is describedlater herein.

Compound 7 or an analog thereof may be converted to a compound ofStructure 4. Essentially, this may be accomplished by protecting the C6and C7 hydroxyl groups and the C17 carbonyl group, and then reducing theΔ⁴ carbon-carbon double bond. Lithium in ammonia/THF is an example of asuitable reducing agent. Such a reduction provides an enolate, which maybe trapped with a suitable electrophile, e.g., trimethylsilyl chlorideor diethylchlorophosphate.

An example of such a conversion is shown in Scheme 2. Thus. protectionof the C6 and C7 hydroxyl groups of compound 7 may be accomplished bytreatment with 2,2-dimethoxypropane and a catalytic amount of(1S)-(+)-10-camphorsulfonic acid (CSA) to produce acetonide 8. The C17carbonyl group of compound 8 may be protected by converting it to anhydroxyl group, and then protecting the hydroxyl group. Chemoselectivereduction of the C17 carbonyl group may be accomplished by use of NaBH₄in methanol to provide compound 9, which in turn is reacted with asuitable hydroxyl protecting group, e.g., t-butyldimethylsilyl chloride,to provide silyl ether compound 10. Compound 10 may be reacted withlithium in liquid ammonia/THF, followed by quenching withdiethylchlorophosphate, to provide compound 11. Compound 11 has a 5αhydrogen, as well as C6 and C7 dihydroxylation, and thus is arepresentative compound of Structure 4.

In an aspect of the present invention, olefinic steroids having anexocyclic olefin at C17 and oxygen atoms at both C6 and C7 are provided.In one embodiment, the olefinic steroid has the Structure 5, includingindividual enantiomeric or geometric isomers thereof, and furtherincluding a solvate or pharmaceutically acceptable salt thereof.Structure 5 is defined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10 and C13 is independently substituted with one of—X, —R⁴ or —OR¹;

C14 is substituted with —X, —OR¹, or —R⁴ excluding methyl;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R², R³ and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

Providing an exocyclic double bond at C17 is readily accomplished by theWittig reaction, starting with a C17 carbonyl compound. Steroids of theinvention having C17 carbonyl functionality are readily available, e.g.,in compound 7 as prepared according to Scheme 1, or by the syntheticsequence summarized in Scheme 3 below, which starts from compound 10 (asprepared in Scheme 2).

Thus, the A-ring of compound 10 can be reduced to afford a C3 carbonylgroup as the only functionality in the A-ring. Scheme 3 illustrates atwo-step sequence to achieve this reduction, wherein compound 10 isreduced with lithium in liquid ammonia and an ether solvent, e.g.,diethyl ether or THF, to provide a mixture of compounds 12 and 13. Thismixture may then be oxidized with a suitable oxidizing agent, forexample PDC, to give exclusively compound 13. Compound 13 may then bereduced with LS-Selectride® (Aldrich Chemical Co., Milwaukee, Wis.) orother selective reducing agent, to provide compound 14 having theindicated stereochemistry.

The 3α-hydroxyl group of compound 14 may then be protected as theacetate using acetic anhydride and pyridine to give compound 15. Othersuitable hydroxyl protecting groups could be used instead of the acetategroup. Removal of the silyl protecting group at C17 can be achievedunder standard conditions known in the art for removing this silylprotecting group, e.g., using tetrabutylammonium fluoride (TBAF), toafford a C17 hydroxyl compound such as compound 16. The C17 hydroxylgroup can be oxidized to a carbonyl group under typical oxidationconditions, e.g., using oxalyl chloride in DMSO and Et₃N, to provideketone compound 17.

Compound 17 can be used in a multitude of olefination reactions,including Wittig-type reactions, to provide compounds of Structure 5having an olefin at C17. For example, compound 17 may be reacted withethyltriphenylphosphonium bromide to provide the ethylidene compound 18.Other starting ketones may be used to provide other steroids having anexocyclic double bond at C17.

As described previously, compounds containing a carbonyl at C17 (orthose that contain functionality that is readily converted to a carbonylgroup) can be transformed into compounds containing a carbon-carbondouble bond at C17 using Wittig chemistry. For example, as outlined inScheme 4 below, compound 19 may be transformed into the correspondingC17 ethylidene compound 23 in a five step process. Thus, the2α,3β-dihydroxy functionality of compound 19 may be protected withhydroxyl protecting groups, (e.g., using 2,2-dimethoxy propane andcamphor sulfonic acid (CSA) in N,N-dimethylformamide (DMF) to give acompound such as compound 20. Deprotection of the C17 hydroxyl may beachieved using reaction conditions suitable for the particular hydroxylprotecting group (in this instance, TBAF in THF may be used) followed byoxidation of the resulting hydroxyl group (e.g., using PDC in CH₂Cl₂)yields the compound containing the C17 ketone (21). Reaction of compound21 with a Wittig reagent, e.g., ethyl triphenyl phosphonium bromide andpotassium t-butoxide in toluene, gives compound 22. Deprotection of thehydroxyl groups in olefin 22 affords the tetrahydroxy compound 23.

Protection steps may be required prior to derivatization at C17 in somecases. For example, in compound 24 (prepared according to Scheme 14) theC3 ketone should first be protected before proceeding with thetransformations at C17 (see Scheme 5 below). Thus, compound 24 may firstbe reduced (e.g., by reaction with NaBH₄ in ethanol) then acylated(e.g., using acetic anhydride in pyridine) to yield the C3,C5-acetoxyderivative 25. Deprotection, oxidation and Wittig chemistry at C17,analogous to that described in Scheme 4 may be used to provide compound27. Subsequent deprotection of the C6 and C7 hydroxyl groups (80% aceticacid is conveniently used to remove the ketal group of compound 27)gives compound 28 which contains the exocyclic Δ¹⁷ olefin.

In an aspect of the present invention, steroids having C17 oxygenationas well as oxygenation at C6 and C7 are provided. In one embodiment, thesteroid has the Structure 6, including individual enantiomeric orgeometric isomers thereof, and further including a solvate orpharmaceutically acceptable salt thereof. Structure 6 is defined asfollows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

R⁵ and R⁶ may together form a direct bond so C17 is a carbonyl group, ormay together with C17 form a cyclic 3–6 membered ether or 4–6 memberedlactone; otherwise R⁵ is R⁴ or —OR⁶ and R⁶ is R¹ or R⁴; and

X represents fluoride, chloride, bromide and iodide.

Preferably, neither the A nor B ring in compounds of Structure 6 isaromatic. In another preferred embodiment, when C10 is substituted withmethyl, then C5 is not directly bonded to an oxygen atom.

Many examples of compounds of Structure 6 and their synthesis havealready been provided above. For instance, compounds 7, 8, 9, 10, 11,13, 14, 15, 16, 17, 19, 20, 21, 24, 25 and 26 are representativecompounds of Structure 6. Many additional compounds of Structure 6,including the synthesis thereof, are provided herein in connection withother compounds of the invention. Therefore, one of ordinary skill inthe art is able to prepare many compounds of Structure 6 in view of thedisclosure herein.

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C1. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C1 for a compound of Structure 6 is providedbelow and outlined in Schemes 6, 7, and 8. It should be recognized thatthe same or analogous synthetic methodology can be applied to provideoxygen and/or hydrocarbon substitution at C1 for any compound ofStructures 5–12 where C1 oxygen and/or hydrocarbon substitution isdesired.

Introduction of an oxygen functionality at C1 of the steroid carbonskeleton can be accomplished by first generating the 1-ene-3-onefunctionalization pattern in the A-ring of a steroid, followed byMichael addition chemistry using any of a number of alkoxide anions, asoutlined in Scheme 6. For example, the enone 29 may be produced fromcompound 13 using standard methodology. The benzyloxy compound 30 maythen be produced by reacting the enone (29) with benzyl alcohol and KOH.Reduction of the C3 ketone of compound 30, and protection of theresultant secondary alcohol as the silyloxy derivative (to providecompound 31) may be followed by catalytic hydrogenation to yield the C1hydroxyl functionality in compound 32. Oxidation of this secondaryalcohol using, e.g., PDC in CH₂Cl₂ may produce compound 33 having a C1ketone.

Compounds containing both an alkyl group and an hydroxyl group at C1 maybe produced by reaction of compound 33 with an alkyl lithium reagent.For example, reaction of compound 33 with CH₃Li in ether will providethe tertiary alcohol in compound 34 (Scheme 7).

Michael addition chemistry similar to that described in Scheme 6 can beused to add an alkyl group to the C1 position. This can be accomplishedusing a number of reagents including R₂CuLi where R may be alkyl, vinylor aryl. For example, compound 29 may be reacted with Me₂CuLi in etherto yield the C1 methyl substituted derivative 35 (Scheme 8).

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C2. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C2 for a compound of Structure 6 is providedbelow. It should be recognized that the same or analogous syntheticmethodology can be applied to provide oxygen and/or hydrocarbonsubstitution at C2 for any compound of Structures 5–12 where C2 oxygenand/or hydrocarbon substitution is desired.

Compounds containing oxygen at C2 may be prepared in a number of waysincluding hydroboration of a silyl enol ether as shown in Scheme 9. Thesilyl enol ether may be prepared from the enone 29 via Li/NH₃ reductionfollowed by trapping of the resultant enolate using TMSCl to yieldcompound 36 (or other R₃SiCl reagents to produce an analogous silyl enolether). Hydroboration of the carbon-carbon double bond in 36 may givethe 2α,3β-dihydroxy functionalization pattern (compound 19). Oxidationof this dihydroxy compound using PDC in CH₂Cl₂ may provide the diketone38.

Preparation of C2 hydrocarbon substituted compounds can be produced,e.g., by α-alkylation of a compound containing a C3 ketonefunctionality. For example, Li/NH₃ reduction of the enone 29 followed bytrapping the resultant anion with an alkylating agent provides C2alkylation. Treatment of the resultant enolate with methyl iodide mayyield the C2 methylated compound 39 (Scheme 10 below). This methodologycan be applied to a variety of different compounds using a number ofdifferent alkyl halides.

Compounds of Structure 6 may have hydrocarbon substitution at C3.Exemplary synthetic methodology to provide hydrocarbon substitution atC3 for a compound of Structure 6 is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide hydrocarbon substitution at C3 for any compound ofStructures 5–12 where C3 hydrocarbon substitution is desired.

Wittig chemistry on compound 13 followed by reduction of the double bondor alternative modifications will provide the alkyl or dialkylderivative at C3. For example, reaction of compound 13 with methyltriphenylphosphonium bromide and tBuOK in toluene may be used to givecompound 40 (Scheme 11). A Simmons-Smith reaction on compound 40 withCH₂I₂ and Zn—Cu followed by catalytic hydrogenolysis of the cyclopropanederivative 41 using H₂, Pd/C in ethanol can be used to give the dialkylderivative 42 (Scheme 11).

Compounds of Structure 6 may have hydrocarbon substitution at C4.Exemplary synthetic methodology to provide hydrocarbon substitution atC4 for a compound of Structure 6 is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide hydrocarbon substitution at C4 for any compound ofStructures 5–12 where C4 hydrocarbon substitution is desired.

Alkylation at C4 may be achieved by first producing the enolate anionfrom the enone in compound 10 (using, for example, reduction withlithium in liquid ammonia) followed by treatment with an appropriatealkyl halide as shown in Scheme 12.

Alternatively, Compounds of Structure 6 may have carbonyl functionalityat C4. Exemplary synthetic methodology to provide carbonyl functionalityat C4 for a compound of Structure 6 is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide carbonyl functionality at C4 for any compound ofStructures 5–12 where a C4 carbonyl group is desired. As describedbelow, the carbonyl functionality at C4 provides a convenient entry intocompounds having a tertiary alcohol and a hydrocarbyl group at C4.

Compounds with a ketone (carbonyl) functionality at C4 may be preparedfrom compound 44 (which in turn is prepared from a deacetylation ofacetate 147 from Scheme 44) by selectively tosylating, epoxidation andthen epoxide ring opening followed by oxidation of the resultant4β-hydroxyl functionality. For example, as illustrated in Scheme 13,treatment of the diol 44 with p-toluenesulfonyl chloride in pyridine andDMF followed by reaction of the resultant tosylate 45 with tBuOK canintroduce the 3β,4β-epoxide (compound 46). Treatment of the epoxide withMe₂CuLi gives the 3α-methyl derivative 47 and subsequent oxidationusing, for example, PDC in CH₂Cl₂ gives the desired ketone (carbonyl) atC4 (compound 48). Epimerization to the 3β-methyl derivative can beachieved using tBuOK in tBuOH and subsequent treatment of the ketonewith a methyl lithium in THF can provide the tertiary alcohol at C4(compound 49).

Alternatively, compounds of Structure 6 may have oxygen or hydrocarbonsubstitution at C5. Exemplary synthetic methodology to provide oxygen orhydrocarbon substitution at C5 for a compound of Structure 6 is providedbelow. It should be recognized that the same or analogous syntheticmethodology can be applied to provide oxygen or hydrocarbon substitutionat C5 for any compound of Structures 5–12 where C5 oxygen or hydrocarbonsubstitution is desired.

Epoxidation of compound 10 followed by ring opening can be used togenerate a hydroxy and subsequently an alkoxy substitution at C5 of thecarbon skeleton. For example, epoxidation of the double bond in compound10 can yield the corresponding epoxide derivative 50 which may bereadily converted to the tertiary hydroxyl compound 24 (Scheme 14below). Subsequent reduction of compound 24 using NaBH₄ in THF andmethylation using MeI in the presence of tBuOK in THF can give thediacetoxy compound 51 (Scheme 15 below). Alkyl substitution at C5 may beachieved using an appropriate alkyl copper lithium reagent. For example,treatment of compound 10 with (CH₃)₂CuLi in ether may produce the C5methyl derivative 52 (Scheme 16).

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C9. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C9 for a compound of Structure 6 is providedbelow. It should be recognized that the same or analogous syntheticmethodology can be applied to provide oxygen and/or hydrocarbonsubstitution at C9 for any compound of Structures 5–12 where C9 oxygenand/or hydrocarbon substitution is desired.

Hydroxylation at the C9 position may be achieved by reaction of aΔ^(9,11) olefinic compound with m-chloroperbenzoic acid followed byreduction with LiAlH₄, as outlined in Scheme 17. For example, using thisprocedure, compound 53 (prepared from the dehydration of compound 60;e.g., NaH, CS₂, MeI, heat) may be used as the starting material toproduce compound 54 which, upon reduction of the epoxide can produce theC9 hydroxyl-containing derivative 55. Subsequent reaction of thetertiary alcohol in compound 55 with dimethyl sulfate in aqueous sodiumhydroxide may be used to give the corresponding alkoxy derivative,compound 56.

Alternatively, compounds of Structure 6 may have hydrocarbonsubstitution at C9. Exemplary synthetic methodology to providehydrocarbon substitution at C9 for a compound of Structure 6 is providedbelow. It should be recognized that the same or analogous syntheticmethodology can be applied to provide hydrocarbon substitution at C9 forany compound of Structures 5–12 where C9 hydrocarbon substitution isdesired.

Cyclopropanation of compound 53 using CH₂I₂ and Zn—Cu followed bycatalytic hydrogenation may provide the corresponding C9-alkylsubstituted compound 57 (Scheme 18).

Alternatively, compounds of Structure 6 may have halide substitution atC9. Exemplary synthetic methodology to provide halide substitution at C9for a compound of Structure 6 is provided below. It should be recognizedthat the same or analogous synthetic methodology can be applied toprovide halide substitution at C9 for any compound of Structures 5–12where C9 halide substitution is desired.

Introduction of a halogen atom at C9 can be achieved in a number of waysincluding reaction of a C9 tertiary alcohol (see, e.g., compound 55 inScheme 17) with thionyl chloride. Thus, reaction of compound 55 withSOCl₂ in CH₂Cl₂, may be used to provide the chloro derivative 59 asshown in Scheme 19.

Compounds of Structure 6 preferably have a methyl substituent at C10.However, the C10 position may be derivatized so as to have manyfunctional groups other than methyl. Exemplary synthetic methodology toprovide oxygen and/or hydrocarbon substitution at C10 for a compound ofStructure 6 is provided below. It should be recognized that the same oranalogous synthetic methodology can be applied to provide oxygen and/orhydrocarbon substitution at C10 for any compound of Structures 5–12where C10 oxygen and/or hydrocarbon substitution is desired.

The derivatization of the C10 position may be achieved via the routeshown in Scheme 20. A 10β-hydroxy steroid 60 (prepared, for example, asoutlined in Scheme 22 below) may be derivatized using nitrosyl chloride(NOCl) in pyridine to yield a nitrite derivative such as 61. Irradiationof the nitrite 61 then can lead to a mixture of the oximes 62 and 63.Compound 63 is reduced to the corresponding imine 64 by treatment withaqueous TiCl₃ in dioxane and acetic acid. The hemiacetal acetate 65 maybe produced upon treatment of 64 with NaNO₂ in aqueous acetic acid. Thiscan also lead to deprotection of the 6,7-hydroxyl groups. The acetonidecan be reintroduced by reaction of the crude product with2,2-dimethoxypropane and camphor sulfonic acid. Alkaline hydrolysis(NaOH, MeOH) to give the hydroxy aldehyde 66 is followed by protectionof the secondary alcohol at C11 as the benzyl ether using BnBr, NaH inDMF to afford compound 67.

A Grignard reaction of compound 67 with CH₃MgBr followed by PDCoxidation in CH₂Cl₂ then by a Bayer-Williger oxidation withm-chloroperbenzoic acid in methylene chloride can give the C10 acetoxyderivative 68. Removal of the acetate group may be accomplished withbase, for example, sodium methoxide in methanol, to give theC10-βalcohol 69. This C10 hydroxyl group may then be further derivatizedto the alkoxide analogue 70, using, for example, sodium hydride in THFfollowed by treatment with an alkylating agent such as methyl iodide.Alternatively, conversion of the C10 hydroxyl group in compound 69 tothe corresponding chloride derivative 71 is achieved using achlorinating agent, e.g., thionyl chloride, as shown in Scheme 21.

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C11. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C11 for a compound of Structure 6 isprovided below. It should be recognized that the same or analogoussynthetic methodology can be applied to provide oxygen and/orhydrocarbon substitution at C11 for any compound of Structures 5–12where C11 oxygen and/or hydrocarbon substitution is desired.

The preparation of compounds of Structure 6 containing an oxygenfunction at the C11 position may be achieved according to the pathwayshown in Scheme 22 from the commercially available starting material 72and related compounds.

If starting with a steroid having a hydroxyl group in the A-ring, as incompound 75 (prepared from the commercially available compound 72(Scheme 22)), removal of the C3 hydroxyl may be achieved using a twostep procedure involving preparation of the methyl xanthate using NaH,CS₂ and CH₃I in THF followed by nBu₃SnH reduction and deprotection (80%AcOH) to yield compound 77. After reduction and protection of the C17ketone using NaBH₄ in methanol followed by TBDMSCl and imidazole in DMF,oxidation of the C7 position can be achieved using a number of oxidizingconditions such as CrO₃ and 3,5-dimethylpyrazole in CH₂Cl₂ or RuCl₃ andtBuOOH in H₂O and cyclohexane. Subsequent reduction (NaBH₄, CeCl₃,THF-MeOH) of the C7 ketone and acetylation can provide the C7 acetoxyderivative 80. Hydroboration of compound 80 provides a product with the6α,7β,11β-hydroxylation pattern as in triol 81. Protection of the 6α,7βhydroxyls in compound 81 using 2,2-dimethoxypropane in the presence ofcamphor sulfonic acid (CSA) followed by oxidation using PDC in CH₂Cl₂gives compound 82 which contains the C11 ketone.

Compounds of Structure 6 may alternatively or additionally havehydrocarbon substitution at C11. Exemplary synthetic methodology toprovide hydrocarbon substitution at C11 for a compound of Structure 6 isprovided below. It should be recognized that the same or analogoussynthetic methodology can be applied to provide hydrocarbon substitutionat C11 for any compound of Structures 5–12 where C11 hydrocarbonsubstitution is desired.

Conversion of the compound 82 C11-ketosteroid to a quaternary alkylcenter may be accomplished as shown in Scheme 23 below.

Thus, the C11-ketosteroid 82 in toluene may be added to a solution ofmethyl triphenylphosphonium bromide and tBuOK to afford compounds with aΔ¹¹ carbon-carbon double bond such as 83. Subsequent treatment of thecompound 83 with CH₂I₂, Zn—Cu may give the cyclopropyl derivative 84.Hydrogenation of the cyclopropane ring (H₂, Pd/C in ethanol) may givethe dialkyl derivative 85. Other Wittig reagents may be employed to makeanalogous alkyl-substituted steroids.

Monoalkylation of the C11 position may be achieved by application ofWittig chemistry on compounds with a C11 ketone, as described above,followed directly by catalytic hydrogenation (as illustrated in Scheme24). For example, catalytic hydrogenation (H₂, Pd/C in ethanol) oncompound 83 affords the C11 methylated steroid 86.

Compounds of Structure 6 may have halide substitution at C11. Exemplarysynthetic methodology to provide halide substitution at C11 for acompound of Structure 6 is provided below. It should be recognized thatthe same or analogous synthetic methodology can be applied to providehalide substitution at C11 for any compound of Structures 5–12 where C11halide substitution is desired.

Thus, halogenation of the C11 position may be achieved according to theroute shown in Scheme 25. For example, treatment of compound 60 with ahalogenating agent, e.g., thionyl chloride in CH₂Cl₂, gives thecorresponding 11β-chloro derivative 87. In general, hydroxylfunctionality may serve as a precursor to halide functionality.

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C12. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C12 for a compound of Structure 6 isprovided below. It should be recognized that the same or analogoussynthetic methodology can be applied to provide oxygen and/orhydrocarbon substitution at C12 for any compound of Structures 5–12where C12 oxygen and/or hydrocarbon substitution is desired.

Placement of an oxygen function at the C12 position may be achieved asillustrated in Scheme 26.

Thus, a C11 ketosteroid, such as compound 82, may be reacted with LDA inTHF followed by trapping of the enolate anion with (Me₂N)₂P(O)Clfollowed by reduction of the enolphosphate using Li and EtNH₂, toprovide a compound, such as 88, with a Δ^(11,12), carbon-carbon doublebond. Epoxidation may be achieved using an epoxidizing agent, e.g.,mCPBA in CH₂Cl₂, to give the corresponding 11α,12α-epoxide derivative89. Subsequent LiAlH₄ reduction of the epoxide can form the 12α-hydroxyderivative (90) which can be oxidized using the appropriate oxidizingagent, for example, pyridinium dichromate (PDC) in methylene chloride,to give the desired C12 ketosteroid 91.

Compounds of Structure 6 may have hydrocarbon substitution at C12.Exemplary synthetic methodology to provide hydrocarbon substitution atC12 for a compound of Structure 6 is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide hydrocarbon substitution at C12 for any compound ofStructures 5–12 where C12 hydrocarbon substitution is desired.

Alkyl groups, such as methyl, may be introduced into the C12 position asshown in Scheme 27 below. The C11 ketosteroid 82, (prepared, forexample, according to Scheme 22), and a strong base, e.g., lithiumdiisopropylamide in THF, are combined and treated with an alkylatingagent, e.g., methyl iodide, to afford the C12 methylated product 92. Atthis stage, the C11 ketone can be removed using a number of methodsincluding those described in connection with Scheme 26 to give themonomethylated product 93. Further treatment with strong base and analkylating agent, e.g., lithium diisopropylamide and methyl iodide,gives the C12 dimethylated product 94. Again, this compound may besubjected to reducing conditions to remove the C11 ketone group thusgiving the C12 dimethyl derivative 95.

Compounds of Structure 6 may have oxygen and hydrocarbon substitution atC12. Exemplary synthetic methodology to provide oxygen and hydrocarbonsubstitution at C12 for a compound of Structure 6 is provided below. Itshould be recognized that the same or analogous synthetic methodologycan be applied to provide oxygen and hydrocarbon substitution at C12 forany compound of Structures 5–12 where C12 oxygen plus hydrocarbonsubstitution is desired.

Scheme 28 shows the preparation of a tertiary alcohol at the C12position from the corresponding C12 ketone. In Scheme 28, the C12 ketone91 is treated with a alkyl lithium reagent, e.g., methyl lithium indiethyl ether, to give the desired tertiary alcohol 96.

Compounds of Structure 6 may have carbon, oxygen or halogen, to name afew atoms, bonded to C13. Exemplary synthetic methodology to providesuch substitution at C13 for a compound of Structure 6 is providedbelow. It should be recognized that the same or analogous syntheticmethodology can be applied to provide the same or analogous substitutionat C13 for any compound of Structures 5–12 where such C13 substitutionis desired.

Substituents at the C13 position may be introduced according to thepathway shown in Scheme 29 below. In a fashion similar to thatpreviously described in Scheme 20, the C13 position can be substitutedwith a hydrocarbyloxy moiety, e.g., a methoxy moiety. Thus, the oximederivative 62, (prepared, for example, as described in Scheme 20), isreduced to the corresponding imine 97 by treatment with aqueous TiCl₃ indioxane and acetic acid. The hemiacetal acetate 98 may be produced upontreatment of compound 97 with NaNO₂ in aqueous acetic acid. Alkalinehydrolysis (NaOH, MeOH) to give the hydroxy aldehyde 99 is followed byprotection of the secondary alcohol at C11 as the benzyl ether usingBnBr, NaH in DMF to afford compound 100.

A Grignard reaction on compound 100 may be used to introduce additionalfunctionality at C13. For example, treatment of compound 100 with methylmagnesium bromide, followed by oxidation of the resulting C13 secondaryalcohol gives the methyl ketone substituent at C13. This may beoxidized, e.g.; using Bayer-Williger oxidation with m-chloroperbenzoicacid in methylene chloride, to give the C13 acetoxy derivative 101. Thisester may be hydrolyzed by treatment with sodium methoxide in methanolto produce the tertiary alcohol 102. Subsequent reaction of the alcoholwith sodium hydride in THF followed by its quenching with methyl iodidemay be used to produce the C13 methoxysteroid 103. Other alkylatingagents could be used to prepare other hydrocarbyloxy derivatives. TheC13 hydroxyl moiety may then be converted to a halide, for example achloride, by the reaction of alcohol 102 with thionyl chloride, thusaffording the C13 chlorosteroid 104, as shown in Scheme 30 below.

Compounds of Structure 6 may have hydrocarbon substitution at C14.Exemplary synthetic methodology to provide hydrocarbon substitution atC14 for a compound of Structure 6 is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide hydrocarbon substitution at C14 for any compound ofStructures 5–12 where C14 hydrocarbon substitution is desired.

For example, introduction of an alkyl group at C14 of the steroid carbonskeleton could be accomplished by alkylation at the C14 position. Oneapproach to achieve such an alkylation is shown in Scheme 31 below.Initially, preparation of the enone 107 can be accomplished bydeprotection (TBAF, THF) of compound 105 followed by oxidation of thesecondary alcohol using PDC in CH₂Cl₂ to give the C17 ketone derivative106. Conversion of the ketone 106 to the enone 107 may be achieved usingisopropenyl acetate and pTsOH to produce the intermediate enol acetatefollowed by production of the enone using reagents set forth in Scheme31. This is followed by conversion of the enone 107 to the silyl enolether 108 by reacting enone 107 with lithium diethylamide in THFfollowed by reaction of the resultant anion with triisopropylsilyltriflate (TIPSOTf). The cyclopropane derivative 109 is then preparedfrom silyl ether 108 using CH₂I₂ and Zn—Cu. Deprotection of the silylenol ether and cleavage of the cyclopropane ring is achieved using TBAFin THF followed by tBuOK in DMSO and aqueous work-up procedures.

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C15. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C15 for a compound of Structure 6 isprovided below. It should be recognized that the same or analogoussynthetic methodology can be applied to provide oxygen and/orhydrocarbon substitution at C15 for any compound of Structures 5–12where C15 oxygen and/or hydrocarbon substitution is desired.

For example, introduction of an oxygen functionality at C15 of thesteroid carbon skeleton could be accomplished by Michael addition typechemistry using any of a number of alkoxide anions. As outlined inScheme 32 below, the 4-methoxybenzyloxy compound 111 (a representativeC15-hydrocarbyloxy steroid derivative of the invention, where the4-methoxybenzyloxy group (MPMO) serves as an hydroxyl protecting group)could be produced by reacting the enone 107 with 4-methoxybenzyl alcoholand base (e.g., powdered KOH). The 4-methoxybenzyl protecting group maybe removed under oxidizing conditions, e.g., by2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation, to yield theC15 hydroxyl group compound 112). When this is followed by oxidation ofthe secondary alcohol (using, for example, PDC in CH₂Cl₂), thecorresponding C15 ketone (compound 113) may be produced.

Compounds containing an alkyl group at C15 may also be produced by aMichael type conjugate addition. For example, reaction of compound 107with an organolithium cuprate (e.g., Me₂CuLi) in Et₂O may be used toproduce the methyl derivative 114 as shown in Scheme 33.

Compounds containing both a hydrocarbyl (e.g., an alkyl) group and ahydrocarbyloxy (e.g., an alkoxy) group at C15 can be produced by usingGrignard chemistry on compound 117, as outlined in Scheme 34 below.Compound 117 may be prepared in a three step process involving thereduction (e.g., nBu₃SnH reduction of a methyl xanthate prepared fromthe C17 hydroxy analog of compound 111) to afford steroid 115, followedby oxidative removal (e.g., using DDQ) of the MPM protecting groupyielding the secondary alcohol derivative 116. Subsequent oxidation ofcompound 116 to the corresponding ketone yields compound 117. A Grignardreaction on compound 117 using an alkylmagnesium bromide reagent (e.g.,CH₃MgBr) in ether produces the tertiary alcohol 118. Methylation of thetertiary alcohol in 118 with an alkylating agent (e.g., CH₃I (note thatan acylating agent can be used in place of an alkylating agent, inScheme 34 and in every Scheme herein having an alkylating agent)) in thepresence of base (e.g., K₂CO₃) yields the tertiary methoxy compound 119.

Compounds of Structure 6 may have oxygen and/or hydrocarbon substitutionat C16. Exemplary synthetic methodology to provide oxygen and/orhydrocarbon substitution at C16 for a compound of Structure 6 isprovided below. It should be recognized that the same or analogoussynthetic methodology can be applied to provide oxygen and/orhydrocarbon substitution at C16 for any compound of Structures 5–12where C16 oxygen and/or hydrocarbon substitution is desired.

Introduction of a tertiary hydroxyl group at C16 of the steroid carbonskeleton may be accomplished using Grignard chemistry on compound 121 asshown in Scheme 35 below. The ketone 121 may be produced viahydroboration of the olefin (using, for example, Sia₂BH in THF thenaqueous NaOH, H₂O₂) of compound 308 (prepared from compound 106 asoutlined in Scheme 35) to afford alcohol 120. The desired C16 ketonefunctionality may then generated by oxidizing the secondary alcohol atC16, using, for example, PDC in CH₂Cl₂, to yield compound 121. Reactionof the ketone 121 with a Grignard reagent, e.g., CH₃MgBr in ether, maybe used to produce the corresponding tertiary alcohol derivative, inthis example, compound 122. The corresponding alkoxy derivative 123could then be produced directly from compound 122 using the appropriatebase and alkyl halide.

Alkoxy groups at C16 may be produced directly from the corresponding C16hydroxyl compound. For example, compound 124 may be produced by reactingcompound 120 with an reagent, e.g., CH₃I, and a base, e.g., K₂CO₃(Scheme 36).

C16 alkyl groups may be introduced by the direct alkylation of compoundsthat contain a C17 carbonyl. For example, reaction of compound 106 withCH₃I and LDA (other strong bases and alkylating agents could be used) inTHF yields the C16 methyl compound 125 (Scheme 37).

Compounds of Structure 6 have oxygen and/or hydrocarbon substitution atC17, including tertiary alcohol and hydroxyl functionality. Exemplarysynthetic methodology to provide tertiary alcohol and hydroxylsubstitution at C17 for a compound of Structure 6 is provided below. Itshould be recognized that the same or analogous synthetic methodologycan be applied to provide tertiary alcohol and hydroxyl substitution atC17 for any compound of Structures 5–12 where C17 tertiary alcohol orhydroxyl substitution is desired.

Thus, Grignard chemistry similar to that described in Scheme 34 may beused to add a tertiary alcohol functionality to the C17 position. Forexample, as outlined in Scheme 38 below, compound 106 may be reactedwith CH₃MgBr in ether to yield the tertiary alcohol derivative 126.Methylation of the resultant tertiary alcohol gives the correspondingC17 methoxy compound 127. Of course, other alkylating agents could beused to provide a wide range of hydrocarbyloxy compounds.

In an aspect of the present invention, C5 stereodefined steroids havinghydroxylation at C6 and C7, a 5α hydrogen and no oxygen atom bonded toC3 is provided. In one embodiment, the stereodefined steroid has theStructure 7, including individual enantiomeric or geometric isomersthereof, and further including a solvate or pharmaceutically acceptablesalt thereof. Structure 7 is defined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C8, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

C3 is substituted with one of ═C(R⁴)(R⁴) and —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)—wherein n ranges from 1 to about 6, or two of —X, and —R⁴;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represent acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur; where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In a preferred embodiment of compounds of Structure 7, C3 is not bondedto an oxygen atom. In another preferred embodiment, when C3 issubstituted with two hydrogen atoms then C17 is not substituted witheither —CH(CH₃)(CH₂)₃CH(CH₃)₂ or —CH(CH₃)(CH₂)2C(═O)OCH₃.

Compounds of Structure 7 have hydroxylation at C6 and C7 and a 5αhydrogen. A synthetic sequence for the preparation of compounds havingthese structural features has been set forth above, in Scheme 2, whichshows the preparation of compound 11. While compound 11 has an oxygenatom bonded to C3, and is thus not a representative compound ofStructure 7, compound 11 can be converted to a compound of Structure 7.Thus, as shown in Scheme 39 below, compound 11 may be reduced tocompound 128, where lithium in liquid ammonia/t-butanol may be used toafford the desired reduction. Hydrogenation of compound 128 can providecompound 105, having a —CH₂— group at C3, as also shown in Scheme 39.

As illustrated in Scheme 40, compound 128 may alternatively be convertedto additional compounds of Structure 7. Thus, the C17 protected hydroxylgroup of compound 128 may be deprotected to yield compound 129, and thenthe C17 hydroxyl group of compound 129 may be oxidized to a C17 carbonylgroup as in compound 106.

Compounds of Structure 7 containing a methylene at C3 may be obtainedfrom compounds with an hydroxyl, protected hydroxyl or ketonefunctionality at C3. The same or analogous synthetic methodology may beused to prepare compounds of any of Structures 5–12 wherein a methylenegroup at C3 is desired.

For example, Scheme 39 above describes the conversion of compound 11 tocompound 105 using chemistry described earlier. Thus, the chemistrydescribed in connection with the Schemes herein can be extended toinclude compounds containing a methylene rather than an hydroxyl orcarbonyl group at C3. In some cases, however, a series of protectionand/or deprotection steps is first required.

Scheme 41 shows an example where the C3 silyloxy functionality mustfirst be deprotected prior to the deoxygenation reaction. The TBDMSgroup in compound 31 may be removed using TBAF in THF. Preparation ofthe methyl xanthate derivative of compound 130 using KH, CS₂ and MeI isfollowed by nBu₃SnH reduction gives the compound (131) containing amethylene group at C3 and a protected hydroxyl group at C1. Oxidation tothe C1 ketone is then achieved by removal of the C1 protecting groupfollowed by using a suitable oxidizing agent, e.g., PDC in CH₂Cl₂, togive compound 132.

Compounds of Structures 5–12, including Structure 7, having C3 alkylfunctionality may be obtained by Wittig chemistry (prepared from the C3ketone as described in connection with Scheme 11). A number of Wittigreagents can be used for this purpose giving rise to substituents withvarious chain lengths and branching.

In an aspect of the present invention, demethylated steroids areprovided which have oxygen and/or hydrocarbon substitution at C6 and C7,however do not have methyl groups at both of C10 and C13. In oneembodiment, the demethylated steroid has the Structure 8, includingindividual enantiomeric or geometric isomers thereof, and furtherincluding a solvate or pharmaceutically acceptable salt thereof.Structure 8 is defined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

with the provisos that (a) C10 and C13 are not simultaneouslysubstituted with methyl, and (b) when C10 is substituted with methyl,then C14 is not substituted with a methyl;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated,

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represent acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur; where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In a preferred embodiment, compounds of Structure 8 do not have aromaticA rings.

A number of examples of compounds containing substituents other thanmethyl at C10 or C13 are described herein in connection with Schemes 20,29 and 30. The substituents include carbonyl, hydroxymethylene, methoxy,ketal, lactone carbonyl, aldehyde, hydroxy, etc. Not described inconnection with Schemes 20, 29 and 30 are compounds containing nosubstitution (i.e., merely hydrogen substitution) at C10 and/or C13.Below are examples discussing synthetic approaches to producing19-nor-6α,7β-dioxygenated steroids.

The synthesis of many of the various compounds of the present inventionhas been detailed in connection with compounds 1 and 247, bothcommercially available starting materials. However, the preparation ofanalogous compounds, e.g., compound 141, that differs only in the lackof a C10 methyl substituent, can be achieved according to Scheme 42shown below.

In Scheme 42, the starting material is the commercially available19-nor-testosterone (133) (Steraloids Inc., Wilton, N.H., or AldrichChemical Company, Milwaukee, Wis.). Reduction of compound 133 usingNaBH₄ in ethanol may give compound 134 which contains the 3β-hydroxylgroup. After protection of the 3β-hydroxyl group using TBDMSCl andimidazole in DMF, allylic oxidation on the resultant diprotectedcompound (135) may be used to afford the enone derivative 136. Reductionand acetylation, as described in previous Sections (Scheme 1), followedby hydroboration using BH₃-THF and oxidative work up (H₂O₂, 30% NaOH),gives compound 138 which contains the 6α,7β,17β-hydroxylation pattern.Protection of the 6α,7β-hydroxyls using 2,2-dimethoxypropane and camphorsulfonic acid can be followed by oxidation of the C17 hydroxyl groupusing PDC in CH₂Cl₂ to afford compound 140 containing the C17 ketonefunctionality. Reaction of compound 140 with the Wittig reagent preparedfrom ethyltriphenylphosphonium bromide and tBuOK in toluene gives theethylidene derivative which can be deprotected in 80% acetic acid toyield the trihydroxy compound 141 which is identical to compound 333except for the lack of a C10 methyl substituent.

In an aspect of the present invention, polyoxygenated steroids havingoxygen and/or hydrocarbon substitution at each of C3, C4, C6 and C7,where the oxygen and/or hydrocarbon substitution at C6 has the alphastereochemistry and the oxygen and/or hydrocarbon substitution at C7 hasthe beta stereochemistry, are provided. In one embodiment, thepolyoxygenated steroid has the Structure 9, including individualenantiomeric or geometric isomers thereof, and further including asolvate or pharmaceutically acceptable salt thereof. Structure 9 isdefined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C11, C12, C15, C16 and C17 is independently substitutedwith

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

with the proviso that C17 is not substituted with any of the following:

each of C5, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

C8 is substituted with —X or —R⁴ and is preferably not bonded directlyto oxygen;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

Compounds having the oxygen and/or hydrocarbon substitution shown inStructure 9 may be prepared from compound 142, which was prepared asdescribed below in Scheme 52. Thus, as shown in Scheme 43, compound 142may be epoxidized with any number of epoxidization conditions, e.g.,using m-chloroperbenzoic acid (m-CPBA) in dichloromethane, to providethe epoxide compound 143. Ring opening of the epoxide group using a mildorganic acid (e.g., anhydrous acetic acid, which is preferred) providescompound 144, which is a representative compound of Structure 9.

From compound 144, many other compounds of Structure 9 may be prepared.For example, as illustrated in Scheme 43, compound 144 may bedeacetylated to provide the tetrahydroxy ketone compound 145. The ketonegroup at C17 may be subject to Wittig chemistry as discussed above, toprovide entry into a large class of tetrahydroxy olefin compounds ofStructure 9.

Structure 9, which has a 3,4,6,7-tetraoxygenation pattern, mayadditionally contain further oxygen-containing substituents. Forexample, compounds of Structure 9 may have an oxygen atom at C11.Synthetic methodology to introduce a C11 oxygen atom, which may beemployed to prepare compounds of Structures 5–12 including Structure 9,may be achieved by chemistry shown in Scheme 44, or by chemistryanalogous to that shown in Scheme 44.

For example, rather than using a commercially available startingmaterial with a C11 hydroxyl functionality or Δ^(9,11) carbon-carbondouble bond, the formation of the m-bischloroiodosobenzylformyl esterfollowed by its photolysis generates the desired unsaturation at theΔ^(9,11) position (compound 149). Thus, the C6 and C7 hydroxyls incompound 146 (prepared according to Scheme 61) may be protected using2,2-dimethoxypropane and camphor sulfonic acid to give compound 147.Subsequent reaction of 147 with m-bischloroiodosobenzylformyl chloridein pyridine followed by photolysis in CCl₄ gives compound 149.Protection of the A-ring hydroxyl groups followed byhydroboration/oxidation yields the C11 hydroxy derivative 151. Completedeprotection using 80% acetic acid gives the hexol 152.

In an aspect of the present invention, steroid ketones having a pyran orδ-lactone ring in the C17 sidechain are provided. In one embodiment, thesteroid ketone has the Structure 10, including individual enantiomericor geometric isomers thereof, and further including a solvate orpharmaceutically acceptable salt thereof. Structure 10 is defined asfollows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

with the proviso that C3 and C4 are not simultaneously substituted withhydroxyl or protected hydroxyl, and are preferably not simultaneouslysubstituted with oxygen atoms;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

G is —C(═O)—, —CH(OR¹)—, —C(R⁴)(OR¹)— or —C(OR¹)(OR¹)—;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

Convenient access to the C17 sidechain in compounds of Structure 10begins with L-carvone, as shown in Scheme 45.

L-Carvone (153) may be converted to compound 154 according to literatureprocedures. See, e.g., Tetrahedron Letters 25(41):4685–4688 (1984). Theprimary alcohol in compound 154 is then protected by, e.g., conversionto an acetate ester. Removal of the ketal protecting group in compound155 using acidic conditions provides aldehyde 156.

The compound 156 can provide access to the C17 sidechain in compounds ofStructure 10 as shown in Scheme 46 below. Thus, compound 145 as preparedin Scheme 43, may be treated with the ylid prepared fromethyltriphenylphosphonium bromide and base to afford compound 157 (usedas starting material in Scheme 46). Thereafter, the four hydroxyl groupsmay be converted to protected hydroxyl groups, for example benzyloxygroups, as shown in compound 158. Compound 158 is then coupled with thealdehyde 156 (Scheme 45) in the presence of a Lewis acid, to providecompound 159. Deprotection of the C29 acetoxy group may then beaccomplished with base, to provide diol compound 160, which may then beoxidized to the δ-lactone compound 161. Allylic oxidation of compound161 may introduce a carbonyl moiety at C15 with concurrent oxidation ofthe benzyl groups (Bn) to benzoate (Bz) groups, to form compound 162.

Reduction of the conjugated Δ¹⁶ carbon-carbon double bond in the D-ringof compound 162 gives compound 163. Removal of the benzoate groups in163 may be achieved using basic conditions (for example NaOMe in MeOH)with concurrent epimerization at C14 to yield product 164 which containsan epimeric mixture of the compounds containing the cis C/D ringjunction and the trans C/D ring junction. Finally, protection of the C15ketone followed by reduction of the δ-lactone to the lactol anddeprotection (80% acetic acid) may be accomplished to give22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (compound 165)and it's C14 epimer22,29-epoxy-3,4,6,7,29-pentahydroxy-14α-stigmastan-15-one.

Compounds of Structure 10 may have a C15 ketone and C22,29 epoxyfunctionality. In fact, compounds containing a variety of functionalityin the A-D rings in addition to a C15 ketone and a sidechain hemiacetalmay be produced using a combination of methodology described herein.

For example, as illustrated in Scheme 47, compound 176, which contains amethylene at C3, a carbonyl at C15 and a sidechain hemiacetal, may beproduced by using methodology described herein. The C15 ketone and thesidechain hemiacetal may then be incorporated using methodologydescribed in detail above (in connection with Schemes 45 and 46).

As shown in Scheme 47, compound 76 can be deprotected using H₂, Pd/C inethanol to give a compound containing the C11 hydroxyl functionalitywhich, upon heating in POCl₃ and pyridine, may produce compound 167containing the Δ^(9,11) double bond and its Δ^(11,12) isomer.Epoxidation (of 167) using mCPBA followed by LiAlH₄ reduction may beused to afford compound 169 which contains the C9 hydroxyl functionalgroup. Protection of this hydroxyl group followed by removal of theketal protecting group and Wittig chemistry may be done to yield theolefinic product 171. Conversion of compound 171 to lactol 176 may beaccomplished using standard methods described herein.

A second example involves the preparation of derivative 186, a compoundthat contains the C15 ketone and the sidechain hemiacetal as well as aC1 hydroxyl functionality. Compound 186 may be produced in a multi-stepprocedure from the commercially available starting material 177 as shownin Scheme 48. The first step involves protection of compound 178 usinge.g., ethylene glycol, pTsOH in benzene. Subsequent Michael additionusing, e.g., benzyl alcohol and potassium hydroxide gives the C1benzyloxy derivative 179. LS-Selectride® reduction of the ketone 179followed by protection of the resultant alcohol as the benzyloxyderivative may be used to give compound 180. The conversion of compound180 to lactol 186 may then be achieved using methods described in Scheme47 and described in detail in other previous examples.

Thus, the methodology described herein may be used to produce compoundswith functionality at carbons in the steroidal ring structure as well asboth the C15 ketone functionality and sidechain hemiacetal.

In a related aspect of the present invention, steroids havingoxygenation at C6 and C7, with a pyran- or δ-lactone-containingsidechain at C17 are provided. In one embodiment, the steroid has theStructure 11, including individual enantiomeric or geometric isomersthereof, and further including a solvate or pharmaceutically acceptablesalt thereof. Structure 11 is defined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted with

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

with the proviso that C3 and C4 are not simultaneously substituted withhydroxyl or protected hydroxyl, and are preferably not simultaneouslysubstituted with oxygen atoms;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

G is —C(═O)—, —CH(OR¹)—, —C(R⁴)(OR¹)—or —C(OR¹)(OR¹)—;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

The preparation of compounds of Structure 11 may be achieved usingmethodology set forth many places herein. For example, compounds 196(Scheme 49) and 207 (Scheme 50) may be synthesized from compounds 30 and55 in multi-step processes. Methods used to convert the C17 silyloxygroup in compound 30 to the olefin 190 are analogous to those describedin detail in previous examples, as are the methods used to convertcompound 190 to compound 196. The same holds true for the conversions ofcompounds 55 to 200 and 200 to 207, respectively.

The chemistry described in Schemes 49 and 50 above are just two examplesof how the methods discussed herein may be applied to produce compoundscontaining a 6,7-dioxygenation pattern and the hemiacetal or δ-lactonesidechain. Thus, the methodology described previously may be used toproduce compounds with functionality at C2, C4, C8, etc.

In an aspect of the present invention, steroid epoxides are provided. Inone embodiment, the steroid epoxide has the Structure 12, includingindividual enantiomeric or geometric isomers thereof, and furtherincluding a solvate or pharmaceutically acceptable salt thereof.Structure 12 is defined as follows:

A compound of the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C11, C12, C15, C16 and C17 is independently substitutedwith

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6; or

(b) two of the following, which are independently selected: —X, —R⁴ and—OR¹;

each of C5, C8, C9, C10, C13 and C14 is independently substituted withone of —X, —R⁴ or —OR¹;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

Preferably, in compounds of Structure 12, C7 does not have carbonylsubstitution when C5 has hydroxy or —OR¹ substitution.

As with the previous examples, introduction of functional groups atvarious positions within the steroid ring structure of compoundscontaining a 3,4-epoxide group of Structure 12 may be achieved usingmethods described herein. For example, as shown in Scheme 51, an oxygenatom may be placed at C9 and/or C11, via epoxidation of Δ^(9,11) doublebond.

Thus, LS-selectride reduction followed by remote oxidation usingreagents described earlier on a compound such as compound 10 may providean olefinic compound 208 (Scheme 51). Transformations to the Δ^(9,11)olefin can be achieved using standard methodology and concurrentreaction of both the Δ^(3,4) and Δ^(9,11) double bonds provide thedesired epoxides at C3–C4 and C9–C11. Oxidation of the C3 hydroxylmoiety with PDC in CH₂Cl₂ then may be used to give the desiredunsaturated A-ring (and optionally ring-opening the epoxide rings willprovide a 3,6,7,9-polyhydroxylated steroid 215).

The introduction of an alkyl group in the C16 position may also beachieved using similar chemistry described above. In the followingexample (Scheme 52), a methyl group is incorporated into this positionfrom the condensation of the D-ring enolate with methyl iodide. Thismethodology is analogous to that described in connection with Scheme 37.As shown in Scheme 52, the alkylated epoxide 218 may be subjected toepoxide-ring-opening conditions to afford a 3,4,6,7-tetrahydroxy steroid220.

Compounds having 6α,7β-hydroxylation pattern have been discussed inprevious sections. Alternatively, compounds containing otherstereochemistries at C6 and C7 may also be produced as discussed in thefollowing section. For example, selective tosylation of compound 221(prepared according to Scheme 37) using pTsCl in pyridine followed bytreatment with potassium carbonate may yield the epoxide containingcompound 223. Subsequent ring opening using aqueous acid may yieldcompounds with the 6β,7α-stereochemistry as shown in Scheme 53.

Compounds with the 6α,7α stereochemistry can be prepared fromcommercially available starting materials as shown in Scheme 54. Thus,cholesteryl acetate may be oxidized using RuCl₃ and tBuOOH in CH₂Cl₂ toafford the enone containing compound 229. Exchange of the protectinggroup at C3 to the tBDMS derivative is followed by lithium ammoniareduction and trapping of the enolate anion with (MeO)₂PCl giving theenol phosphate 231. A second lithium-ammonia reduction gives the Δ^(6,7)double bond which may be oxidized with OsO₄ to afford compound 233containing the 3β,6α,7α-trihydroxylation pattern.

More generally, compounds of the present invention may be characterizedby the following formula:

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

C17 is substituted according to any of (c), (d), (e), (f), (g), (h) and(i):

(c) ═C(R²)(R³) except when C14 is substituted with methyl;

(d) —R⁵ and —OR⁶ so long as the A and B rings are not aromatic, and whenC10 is substituted with methyl then C5 is not bonded directly to oxygen,where R⁵ and R⁶ may together form a direct bond so C17 is a carbonylgroup, or may together with C17 form a cyclic 3–6 membered ether or 4–6membered lactone; otherwise R⁵ is R⁴or —OR⁶ and R⁶ is R¹ or R⁴.

(e) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6, as long as oneof the following conditions i), ii), iii) or iv) apply:

-   -   i) C5 is substituted with a hydrogen in the alpha configuration,        and C3 is not bonded to oxygen, and when C3 is substituted with        two hydrogen atoms then C17 is not substituted with either        —CH(CH₃)(CH₂)₃CH(CH₃)₂ or —CH(CH₃)(CH₂)2C(═O)OCH₃;    -   ii) C10 and C13 are not simultaneously substituted with methyl,        and when C10 is substituted with methyl, then C14 is not        substituted with a methyl, and the A ring is never aromatic;    -   iii) if C3 and C4 are bonded to oxygen atoms. and the C6 —OR¹        substituent has the alpha configuration, and the C7 —OR¹        substituent has the beta configuration, then C17 is not        substituted with any of the following:

-   -   iv) C3 and C4 are each bonded to the same oxygen atom so as to        form an oxirane ring, with the proviso that C7 does not have        carbonyl substitution when C5 has hydroxyl or —OR¹ substitution;

(f) two of the following substituents, which are independently selected:—X, —R⁴ and —OR¹, as long as one of the above conditions i), ii), iii)or iv) apply;

(g) a cyclic structure of the formula

wherein G is —C(═O)—, —CH(OR¹)—, —C(R⁴)(OR¹)— or —C(OR¹)(OR¹)—, as longas C3 and C4 are not simultaneously substituted with hydroxyl orprotected hydroxyl;

(h) two hydrogen atoms, as long as C3 is not substituted with a carbonylgroup;

(i) one hydrogen atom and one group selected from C₁–C₃₀ hydrocarbylgroups and C₁–C₃₀ halogen substituted hydrocarbyl groups, excluding—CH(CH₃)(CH₂)₃CH(CH₃)₂;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R², R³ and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In a preferred embodiment, the compounds of the invention have one ofthe structure set forth below, and mixtures thereof:

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C1, C2, C3, C4, C11, C12 and C16 is independently substitutedaccording to (a) or (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6,

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

C5 is substituted with a hydrogen atom;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹, although C5 is preferablysubstituted with hydrogen; and

C17 is substituted according to (c), (d), (e) or (f):

(c) two substituents selected from hydrogen, halogen, C₁–C₃₀ saturatedhydrocarbyl excluding —CH(CH₃)(CH₂)₃CH(CH₃)₂, halogen substituted C₁–C₃₀saturated hydrocarbyl, C₁–C₃₀ unsaturated hydrocarbyl, and halogensubstituted C₁–C₃₀ unsaturated hydrocarbyl;

(d) one substituent selected from ═C(R⁴)(R⁴) with the proviso that C14is not substituted with methyl;

(e) at least one oxygen atom-containing substituent selected from ═O,—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6, —OH, and —OR¹;

(f) at least one nitrogen atom-containing substituent selected from—N(R⁴)(R⁴) wherein the two R⁴ groups may together with the nitrogen atomform one or more rings, so that the nitrogen atom-containing substituentincludes nitrogen atom-containing heterocyclic groups; wherein

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated, however fully saturated rings arepreferred;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where —OR¹ groups bonded to adjacent carbon atoms may togetherform a cyclic structure which protects both hydroxyl groups;

R⁴ at each occurrence is independently selected from H and R⁵;

R⁵ is a C₁₋₃₀ organic moiety that may optionally contain at least oneheteroatom selected from the group consisting of boron, halogen,nitrogen, oxygen, silicon and sulfur; where two geminal R⁵ groups maytogether form a ring with the carbon atom to which they are both bonded;and

X represents fluoride, chloride, bromide or iodide.

The compounds of general and preferred structures as disclosed hereinmay be prepared by synthetic methodology as set forth in the Schemes1–54, the references cited herein and the Examples provided herein, aswell as knowledge of the skilled artisan. The following are preferredsynthetic procedures useful in preparing compounds of the presentinvention.

In one aspect, the invention provides a process for introducing anexocyclic olefin group to the C17 position of a 6,7-dioxygenatedsteroid. The process includes the step of providing a compound ofFormula (10) (such a compounds may be commercially available or may beprepared by techniques disclosed herein), and then reacting the compoundof Formula (10) with a Wittig reagent of Formula (11) in the presence ofa base, to provide an olefin compound of Formula (12)

Each of the compounds of Formulas (10) and (12) include pharmaceuticallyacceptable salts and solvates thereof. In Formula (10), (11) and (12):

each of C1, C2, C3, C4, C11, C12, C15 and C16 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

Ra, Rb and R⁴ at each occurrence is independently selected from H andC₁₋₃₀ organic moiety that may optionally contain at least one heteroatomselected from the group consisting of boron, halogen, nitrogen, oxygen,silicon and sulfur, where two geminal R⁴ groups may together form a ringwith the carbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide, which isindependently selected at each occurrence.

In preferred embodiments of the process, the base is selected fromsodium t-butoxide, potassium t-butoxide and sodium hydride and the like.The base is preferably in admixture with an aprotic solvent. Suitableaprotic solvents include toluene, tetrahydrofuran, methylene chloride,dimethylformamide, dimethylsulfoxide, benzene and diethyl ether. Inanother preferred embodiment, Ra and Rb are independently selected fromhydrogen and C₁–C₇alkyl, and X is selected from chloride, bromide andiodide.

In another aspect, the invention provides a process for introducing6α,7β-dioxygenation into a steroid. The process includes the steps ofproviding a steroid of Formula (13) having a carbonyl group at C7 and adouble bond between C5 and C6. Steroids of Formula (13) may be preparedby, for example, synthetic methodology disclosed herein. In a subsequentstep, the carbonyl group is reduced to a hydroxyl group, followed by ahydroboration of the double bond to provide a hydroxyl group at C6,wherein the C6 hydroxyl group has the a-configuration and the C7hydroxyl group has the β-configuration,

The compounds of Formulas (13) and (14) include pharmaceuticallyacceptable salts and solvates thereof. In Formula (13) and (14):

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C8, C9, C10, C13 and C14 is independently substituted with oneof —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In preferred embodiments of the process, the reduction is accomplishedwith sodium borohydride in combination with cerium(III) chlorideheptahydrate. In another preferred embodiment of the process, thehydroboration is conducted with a hydroboration reagent selected fromBH₃ and 9-BBN, and preferably in the presence of an aprotic solvent.Suitable aprotic solvents include tetrahydrofuran, methylene chloride,diethyl ether, dimethyl sulfide and carbon disulfide. The hydroborationis preferably immediately followed by treatment with a peroxide, such ashydrogen peroxide or t-butylperoxide, and a base, such as sodiumhydroxide and potassium hydroxide.

Another aspect of the invention provides a process for astereocontrolled introduction of a hydroxyl group at C3 of a steroidnucleus. The process includes the step of providing a steroid compoundof Formula (15) having a carbonyl group at C3. Steroid compounds ofFormula (15) may be prepared by, for example, synthetic methodsdisclosed herein. This is followed by reducing the carbonyl group to ahydroxyl group with a reducing agent so as to provide at least onecompound of Formulas (16) and (17)

Each of the compounds of Formulas (15), (16) and (17) includepharmaceutically acceptable salts and solvates thereof. In the compoundsof Formulas (15), (16) and (17):

each of C1, C2, C4, C11, C12, C15, C16 and C17 is independentlysubstituted according to any of (a) and (b):

(a) one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)— wherein n ranges from 1 to about 6;

(b) two of: —X, —R⁴ and —OR¹, each independently selected;

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with one of —X, —R⁴ or —OR¹;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where vicinal —OR¹ groups may together form a cyclic structurewhich protects vicinal hydroxyl groups, and where geminal —OR¹ groupsmay together form a cyclic structure which protects a carbonyl group,with the proviso that either or both of —OR¹ at C6 and C7 represents acarbonyl or protected carbonyl group;

R⁴ at each occurrence is independently selected from H and C₁₋₃₀ organicmoiety that may optionally contain at least one heteroatom selected fromthe group consisting of boron, halogen, nitrogen, oxygen, silicon andsulfur, where two geminal R⁴ groups may together form a ring with thecarbon atom to which they are both bonded; and

X represents fluoride, chloride, bromide and iodide.

In a preferred embodiment of this process, the reducing agent isselected from lithium trisiamylborohydride, lithiumtri-sec-butylborohydride and potassium tri-sec-butylborohydride, andwill predominantly provide the hydroxyl compound of Formula (16)(relative to the hydroxyl compound of Formula (17)). In anotherpreferred embodiment, the reducing agent is selected from sodiumborohydride and lithium aluminum hydride, and will predominantly providethe hydroxyl compound of Formula (17). In general, the inventive processwill achieve a reduction of compounds of Formula (15) such that theproduct mixture contains a ratio of Formula (16) to Formula (17)compounds of other than 1:1.

As used herein, the term organic moiety of an indicated carbon numberrange refers to a stable arrangement of atoms composed of at least oneand not more than about the maximum carbon number set forth in therange, typically not more than about 30 carbon atoms, and any number ofnon-carbon atoms.

The C₁₋₃₀ organic moiety may be a saturated or unsaturated hydrocarbylradical. A saturated hydrocarbyl radical is defined according to thepresent invention as any radical composed exclusively of carbon andhydrogen, where single bonds are exclusively used to join carbon atomstogether. Thus, any stable arrangement of carbon and hydrogen atoms,having at least one carbon atom, is included within the scope of asaturated hydrocarbon radical according to the invention. Some specificterminology that may be used to refer to specific carbon atomarrangements will be discussed below.

The carbon atoms may form an alkyl group, i.e., an acyclic chain ofcarbon atoms which may be branched or unbranched (linear). Methyl,ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butylare alkyl groups having 1 to 4 carbon atoms (commonly referred to aslower alkyl groups), and are exemplary of alkyl groups of the invention.The carbon atoms may form a cycloalkyl group, i.e., a cyclic arrangementof carbon atoms, where cyclopropyl, cyclobutyl, cyclopentyl arecycloalkyl groups of the invention having 3–5 carbon atoms. Additionalgroups within the scope of “cycloalkyl” as defined herein arepolycycloalkyl groups, defined below.

A polycycloalkyl group is an arrangement of carbon atoms wherein atleast one carbon atom is a part of at least two separately identifiablerings. The polycycloalkyl group may contain bridging between two carbonatoms, where bicyclo[1.1.0]butyl, bicyclo[3.2.1]octyl,bicyclo[5.2.0]nonyl, tricycl[2.2.1.0¹]heptyl, norbornyl and pinanyl arerepresentative examples. The polycycloalkyl group may contain one ormore fused ring systems, where decalinyl (radical from decalin) andperhydroanthracenyl are representative examples. The polycycloalkylgroup may contain a spiro union, in which a single atom is the onlycommon member of two rings. Spiro[3.4]octyl, spiro[3.3]heptyl andspiro[4.5]decyl are representative examples.

In addition, the saturated hydrocarbyl radical can be composed of anycombination of two or more of the above, i.e., any combination of alkyland cycloalkyl groups. Thus, the R⁴ or R⁵ groups may be an alkyl group(e.g., methyl) with a cycloalkyl (e.g., cyclohexyl) substituent, so thatR⁴ or R⁵ is a cyclohexylmethyl group. As another example, R⁴ or R⁵ maybe a cycloalkyl group (e.g., cyclooctyl) having two alkyl substituents(e.g., a methyl and ethyl substituent), so that R⁴ or R⁵ is amethylethylcyclooctyl group. As a final example, R⁴ or R⁵ may be acycloalkyl group with an alkyl substituent, where the alkyl substituentis substituted with a polycycloalkly substituent.

As indicated above, R⁴ or R⁵ may be an unsaturated hydrocarbyl radical.Such an R⁴ or R⁵ group is defined as having a carbon arrangement as setforth above for saturated hydrocarbyl radicals, with the additionalfeature that at least one bond between any two carbon atoms is otherthan a single bond. An alkyl group with a single double bond is referredto as an alkenyl group, while an alkyl group having more than one doublebond is referred to as an alkapolyenyl group, where alkadienyl (2 doublebonds) and alkatrienyl (3 double bonds) are exemplary. An alkyl groupwith a single triple bond is referred to as an alkynyl group, while analkyl group having more than one triple bond is referred to as aalkapolyynyl group, where alkydiynyl (2 triple bonds) and alkatriynyl (3triple bonds) are exemplary.

Likewise, the cycloalkyl group may have one or more double or triplebonds, and be included within the scope of an unsaturated hydrocarbylradical according to the invention. Cycloalkenyl and cycloalkynyl aregeneral names given to groups having a single carbon-based ring with asingle double and triple bond in the ring, respectively. Cycloalkadienylgroups are cycloalkyl groups with two double bonds contained in the ringstructure. The double bond may be exocyclic to the ring, e.g., a carbonatom of the ring may have a ═CH₂ group (i.e., a methylidene group) orhigher homologue bonded to it.

A ring may be unsaturated to the extent of being aromatic, and still beincluded within the scope of an unsaturated hydrocarbyl radical. Thus,an aryl group, for example, phenyl and naphthyl, are included within thescope of such hydrocarbyl groups. As any combination of the above isalso included within the scope of an unsaturated hydrocarbyl radical,aralkyl (R⁴ or R⁵ is an alkyl group with at least one aryl substituent,e.g., benzyl) and alkylaryl (R⁴ or R⁵ is an aryl ring with at least onealkyl substituent, e.g., tolyl) groups are included within the scope ofR⁴ or R⁵. C₆ aryls are a preferred component of organic moieties of theinvention.

R⁴ or R⁵ includes organic moieties that contain a heteroatom.Heteroatoms according to the invention are any atom other than carbonand hydrogen. A preferred class of heteroatoms are naturally occurringatoms (other than carbon and hydrogen). Another preferred class arenon-metallic (other than carbon and hydrogen). Another preferred classconsists of boron, nitrogen, oxygen, silicon, phosphorous, sulfur,selenium and halogen (i.e., fluorine, chlorine, bromine and iodine.,with fluorine and chlorine being preferred). Another preferred classconsists of nitrogen, oxygen, sulfur and halogen. Another preferredclass consists of nitrogen, oxygen and sulfur. Oxygen is a preferredheteroatom. Nitrogen is a preferred heteroatom.

For example, R⁴ or R⁵ may be a hydrocarbyl radical as defined above,with at least one substituent containing at least one heteroatom. Inthis paragraph, R⁴ will be used to refer to both R⁴ and R⁵. In otherwords, R⁴ may be a hydrocarbyl radical as defined above, wherein atleast one hydrogen atom is replaced with a heteroatom. For example, ifthe heteroatom is oxygen, the substituent may be a carbonyl group, i.e.,two hydrogens on a single carbon atom are replaced by an oxygen, to formeither a ketone or aldehyde group. Alternatively, one hydrogen may bereplaced by an oxygen atom, in the form of an hydroxy, alkoxy, aryloxy,aralkyloxy, alkylaryloxy (where alkoxy, aryloxy, aralkyloxy,alkylaryloxy may be collectively referred to as hydrocarbyloxy),heteroaryloxy, —OC(O)R⁴, ketal, acetal, hemiketal, hemiacetal, epoxy and—OSO₃M. The heteroatom may be a halogen. The heteroatom may be anitrogen, where the nitrogen forms part of an amino (—NH₂, —NHR⁴,—N(R⁴)₂), alkylamido, arylamido, arylalkylamido, alkylarylamido, nitro,—N(R⁴)SO₃M or aminocarbonylamide group. The heteroatom may be a sulfur,where the sulfur forms part of a thiol, thiocarbonyl, —SO₃M, sulfonyl,sulfonamide or sulfonhydrazide group. The heteroatom may be part of acarbon-containing substituent such as formyl, cyano, —C(O)OR⁴, —C(O)OM,—C(O)R⁴, —C(O)N(R⁴)₂, carbamate, carbohydrazide and carbohydroxamicacid.

In the above exemplary heteroatom-containing substituents, M representsproton or a metal ion. Preferred metal ions, in combination with acounterion, form physiologically tolerated salts. A preferred metal fromwhich a metal ion may be formed include an alkali metal [for example,lithium (Li), sodium (Na), potassium (K), rubidium (Rb) and cesium (Cs)]an alkaline earth metal [(for example, magnesium (Mg), calcium (Ca) andstrontium (Sr)], or manganese (Mn), iron (Fe), zinc (Zn) or silver (Ag).An alkali metal or an alkaline earth metal are preferred M groups.Sodium, potassium, magnesium and calcium are preferred M groups. Sodiumand potassium are preferred M groups.

Another class of organic moieties according to the invention arehydrocarbyl radicals as defined above, wherein at least one carbon issubstituted for at least one heteroatom. Examples of such organicmoieties are heterocycloalkyl (a cycloalkyl group having at least onecarbon replaced with at least one heteroatom), heterocycloalkenyl,heteroaryl, heteroaryloxy, heteroaralkyl, heteroaralkenyl, etc.Collectively, this class of organic moieties may be referred to asheterohydrocarbyls. Another example of such organic moieties have aheteroatom bridging (a) the radical to which the organic moiety isbonded and (b) the remainder of the organic moiety. Examples includealkoxy, aryloxy, arylalkyloxy and alkylaryloxy radicals, which maycollectively be referred to herein as hydrocarbyloxy radicals ormoieties. Thus, —OR⁴ is an exemplary R⁴ group of the invention. Anotherexample is —NHR⁴.

Examples of heterocycloalkylene are pyrrolidinylene, piperidinylene,tetrahydrofuranylene, di and tetrahydropyranylene. Examples ofheterocycloalkyl are radicals derived from pyrrolidine, imidazolidine,oxazolidine, pyrazolidine, piperidine, piperazine and morpholine.Examples of heterocycloalkenyl substituents are radicals derived byremoval of a hydrogen from 2- and 3-pyrroline, oxazoline, 2- and4-imidazoline and 2- and 3-pyrazoline.

While the organic moiety may have up to about 30 carbon atoms, preferredorganic moieties of the invention have fewer than 30 carbon atoms, forexample, up to about 25 carbon atoms, more preferably up to about 20carbon atoms. The organic moiety may have up to about 15 carbon atoms,or up to about 12 or 10 carbon atoms. A preferred category of organicmoieties has up to about 8 or 6 carbon atoms.

The following are exemplary R⁴ and R⁵ organic moieties where R⁴ or R⁵ isjoined to the steroid nucleus through a carbon atom: alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl, heterocyclylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl, aryloxycarbonyl,heterocyclyloxycarbonyl, carboxylic acid, cyano and formyl.

The following are exemplary R⁴ and R⁵ organic moieties where R⁴ or R⁵ isjoined to the steroid nucleus through an oxygen atom: hydroxy, oxo,alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy,alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy, arylcarbonyloxy andheterocyclyloxy.

R⁴ and R⁵ organic moieties may contain a nitrogen atom through which theR⁴ or R⁵ organic moiety is joined to the steroid nucleus. Examples arenitro and organic moieties of the formula —NL²L³ wherein L² and L³ areindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, aryl, formyl, heterocyclyl, alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl and heterocyclylcarbonyl, such thatL² and L³ together may be alkylene or alkenylene to thereby form a 3- to8-membered saturated or unsaturated ring in combination with thenitrogen atom to which they are attached.

The following are exemplary R⁴ and R⁵ organic moieties where the R⁴ orR⁵ moiety is joined to the steroid nucleus through a sulfur atom:alkylsulfide, alkenylsulfide, alkynylsulfide, cycloalkylsulfide,cycloalkenylsulfide, arylsulfide, heterocyclylsulfide,alkylcarbonylsulfide, alkenylcarbonylsulfide, alkynylcarbonylsulfide,cycloalkylcarbonylsulfide, cycloalkenylcarbonylsulfide,arylcarbonylsulfide, heterocyclylcarbonylsulfide, and groups of theformulas: —S(O)_(n)H, —S(O)_(n)L⁴, —S(O)_(m)OH, —S(O)_(m)OL⁴,—OS(O)_(m)OL⁴, and —O(S)_(m)OH, wherein L⁴ is selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl and heterocyclyl.

In the above R⁴ and R⁵ organic moieties, the alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl and aryl groups (collectively referred to asthe R⁴ or R⁵ hydrocarbyl groups) may be fully or partially halogenated,and/or substituted with up to five L⁵ groups. The heterocyclyl,heterocyclyloxy, heterocyclylcarbonyl, heterocyclyloxycarbonyl,heterocyclylcarbonyloxy groups (collectively referred to as the R⁴heterocyclyl groups) may likewise be fully or partially halogenatedand/or substituted with up to five L⁵ groups.

L⁵ groups contain a carbon, oxygen, nitrogen or sulfur atom throughwhich they are joined to a carbon atom of the R⁴ or R⁵ hydrocarbylgroups or a carbon or nitrogen atom of the R⁴ or R⁵ heterocyclyl groups.

The following are exemplary L⁵ groups wherein a carbon atom of L⁵ isjoined to the R⁴ hydrocarbyl or heterocyclyl group: alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, alkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, arylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl and aryloxycarbonyl.

The following are exemplary L⁵ groups wherein an oxygen atom of L⁵ isjoined to the R⁴ hydrocarbyl or heterocyclyl group: hydroxy, oxo,alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy,alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy and arylcarbonyloxy.

The L⁵ group may contain a nitrogen atom through which the L⁵ group isjoined to the R⁴ or R⁵ hydrocarbyl or heterocyclyl group. Examplesinclude nitro and nitrogen-containing groups of the formula —NL⁶L⁷wherein L⁶ and L⁷ are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, formyl, alkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl andarylcarbonyl such that L⁶ and L⁷ together may be alkylene or alkenyleneto thereby form a 3- to 8-membered saturated or unsaturated ring incombination with the nitrogen atom to which they are attached.

The following are exemplary L⁵ groups wherein a sulfur atom of L⁵ isjoined to the R⁴ or R⁵ hydrocarbyl or heterocyclyl group: alkylsulfide,alkenylsulfide, alkynylsulfide, cycloalkylsulfide, cycloalkenylsulfidearylsulfide, alkylcarbonylsulfide, alkenylcarbonylsulfide,alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,cycloalkenylcarbonylsulfide, arylcarbonylsulfide, and groups of theformulas: —S(O)_(n)L⁸, —S(O)_(m)OH, —S(O)_(m)OL⁸, —OS(O)_(m)OL⁸, and—O(S)_(m)OH, wherein L⁸ is selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl and heterocyclyl.

In the exemplary R⁴ and R⁵ organic moieties, the alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl and aryl groups which form part of L⁵(collectively referred to as the L⁵ hydrocarbyl groups) may be fully orpartially halogenated, and/or substituted with up to three L⁹ groups.The heterocyclyl, heterocyclyloxy, heterocyclylcarbonyl,heterocyclyloxycarbonyl, heterocyclylcarbonyloxy groups (collectivelyreferred to as the L⁵ heterocyclyl groups) may likewise be fully orpartially halogenated, and/or substituted with up to three L⁹ groups.

L⁹ groups contain a carbon, oxygen, nitrogen or sulfur atom throughwhich they are joined to the L⁵ hydrocarbyl group or the L⁵ heterocyclylgroup.

The following are exemplary L⁹ groups wherein a carbon atom of L⁹ isjoined to the L⁵ hydrocarbyl or heterocyclyl group: alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl, heterocyclylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl, aryloxycarbonyl andheterocyclyloxycarbonyl.

The following are exemplary L⁹ groups wherein an oxygen atom of L⁹ isjoined to the L⁵ hydrocarbyl or heterocyclyl group: hydroxy, oxo,alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy,heterocyclyloxy, alkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy,arylcarbonyloxy and heterocyclylcarbonyloxy.

The L⁹ group may contain a nitrogen atom through which the L⁹ group isjoined to the L⁵ hydrocarbyl or heterocyclyl group. Suchnitrogen-containing L⁹ groups include nitro and groups having theformula —NL¹⁰L¹¹ wherein L¹⁰ and L¹¹ are independently hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, formyl,alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl and heterocyclylcarbonyl such thatL¹⁰ and L¹¹ together may be alkylene or alkenylene to thereby form a 3-to 8-membered saturated or unsaturated ring in combination with thenitrogen atom to which they are attached.

The following are exemplary L⁹ groups wherein an sulfur atom of L⁹ isjoined to the L⁵ hydrocarbyl or heterocyclyl group: alkylsulfide,alkenylsulfide, alkynylsulfide, cycloalkylsulfide, cycloalkenylsulfide,arylsulfide, heterocyclylsulfide alkylcarbonylsulfide,alkenylcarbonylsulfide, alkynylcarbonylsulfide,cycloalkylcarbonylsulfide, cycloalkenylcarbonylsulfide,arylcarbonylsulfide, heterocyclylcarbonylsulfide and groups of theformulas: —S(O)_(n)L¹², —S(O)_(m)OH, —S(O)_(m)OL¹², —OS(O)_(m)OL¹², and—O(S)_(m)OH, wherein L¹² is selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl and heterocyclyl.

In the exemplary R⁴ and R⁵ organic moieties, the alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl and aryl groups which form part of L⁹(collectively referred to as the L⁹ hydrocarbyl groups) may be fully orpartially halogenated, and/or substituted with up to three L¹³ groups.The heterocyclyl, heterocyclyloxy, heterocyclylcarbonyl,heterocyclyloxycarbonyl, heterocyclylcarbonyloxy groups (collectivelyreferred to as the L⁹ heterocyclyl groups) may likewise be fully orpartially halogenated, and/or substituted with up to three L¹³ groups.

An L¹³ group contains a carbon, oxygen, nitrogen or sulfur atom throughwhich the L¹³ group is joined to the L⁹ hydrocarbyl group or L⁹heterocyclyl group.

The following are exemplary L¹³ groups wherein a carbon atom of L¹³ isjoined to the L⁹ hydrocarbyl or heterocyclyl group: alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl, heterocyclylcarbonyl,alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl, aryloxycarbonyl andheterocyclyloxycarbonyl.

The following are exemplary L¹³ groups wherein an oxygen atom of L¹³ isjoined to the L⁹ hydrocarbyl or heterocyclyl group: hydroxy, oxo,alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy,heterocyclyloxy, alkylcarbonyloxy, alkenylcarbonyloxy,alkynylcarbonyloxy, cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy,arylcarbonyloxy and heterocyclylcarbonyloxy.

The L¹³ group may contain a nitrogen atom through which the L¹³ group isjoined to the L⁹ hydrocarbyl or heterocyclyl group. Suchnitrogen-containing L¹³ groups include nitro and groups of the formula—NL¹⁴L¹⁵ wherein L¹⁴ and L¹⁵ are independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, formyl,alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, arylcarbonyl and heterocyclylcarbonyl such thatL¹⁵ and L¹⁵ together may be alkylene or alkenylene to thereby form a 3-to 8-membered saturated or unsaturated ring in combination with thenitrogen atom to which they are attached.

The following are exemplary L¹³ groups wherein an sulfur atom of L¹³ isjoined to the L⁹ hydrocarbyl or heterocyclyl group: alkylsulfide,alkenylsulfide, alkynylsulfide, cycloalkylsulfide, cycloalkenylsulfide,arylsulfide, heterocyclylsulfide, alkylcarbonylsulfide,alkenylcarbonylsulfide, alkynylcarbonylsulfide,cycloalkylcarbonylsulfide, cycloalkenylcarbonylsulfide,arylcarbonylsulfide, heterocyclylcarbonylsulfide and groups of theformulas: —S(O)_(n)L¹⁴, —S(O)_(m)OH, —S(O)_(m)OL¹⁴, —OS(O)_(m)OL¹⁴, and—O(S)_(m)OH, wherein L¹⁴ is selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl and heterocyclyl.

In the groups set forth above, m is independently 1 or 2, and n isindependently 0, 1 or 2.

Certain of the R⁴ and R⁵ substituents may contain asymmetric carbonatoms. Compounds containing such substituents may therefore exist inenantiomeric and diastereomeric forms and in racemic mixtures thereof.All are within the scope of the present invention. A racemate or racemicmixture does not imply a 50:50 mixture of stereoisomers.

In accordance with the description of exemplary R⁴ or R⁵ organicmoieties, the following terms have the designated meanings, unlessexplicitly stated otherwise:

Alkyl, alkenyl and alkynyl refer to straight or branched chainhydrocarbons having 1 to 30 carbon atoms (at least two carbon atoms foran alkynyl group) and no unsaturation, at least one double bond or atleast one triple bond, respectively. Preferred carbon number ranges are1 to 20 and 1 to 10.

Cycloalkyl and cycloalkenyl refer to cyclic hydrocarbon groups of 3 to 8carbon atoms, where a cycloalkyl group is saturated, and a cycloalkenylgroup has at least one double bond within the cyclic structure. Suitablecycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl.

Aryl refers to refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups.

Carbocyclic aryl refers to aromatic groups wherein the ring atoms of thearomatic ring are carbon atoms. Carbocyclic aryl groups include phenyl,naphthyl and indenyl groups.

Heterocyclic aryl refers to a mono- or bicyclic ring system of about 5to about 12 carbon atoms, where each monocyclic ring may possess from 0to about 4 heteroatoms, and each bicyclic ring may possess about 0 toabout 5 heteroatoms selected from N, O, and S provided said heteroatomsare not vicinal oxygen and/or sulfur atoms. Examples of such mono- andbicyclic ring systems include, without limitation, benzofuran,benzothiophene, indole, benzopyrazole, coumarin, isoquinoline, pyrrole,thiophene, furan, thiazole, imidazole, pyrazole, triazole, quinoline,pyrimidine, pyridine, pyridone, pyrazine, pyridazine, isothiazole,isoxazole and tetrazole.

Biaryl refers to phenyl substituted by carbocyclic aryl or heterocyclicaryl as defined herein, ortho, meta or para to the point of attachmentof the phenyl ring.

Heterocyclyl refers to a stable 5- to 7-membered mono- or bicyclic orstable 7- to 10-membered bicyclic heterocyclic ring system any ring ofwhich may be saturated or unsaturated, and which consists of carbonatoms and from one to three heteroatoms selected from the groupconsisting of N, O and S, and wherein the nitrogen and sulfurheteroatoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized, and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring. Theheterocyclic ring may be attached to the steroid nucleus through anyheteroatom or carbon atom of the heterocyclic ring which results in thecreation of a stable structure. Examples of such heterocyclic groupsinclude piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolodinyl, 2-oxcazepinyl, azepinyl, pyrrolyl, 4-piperidonyl,pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl,benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl,thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide,thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same asmorpholinyl.

Heterocyclyloxy and heterocyclylcarbonyl refer to heterocyclyl groupsbonded through an oxygen atom or a carbonyl group, respectively, to oneof the to one or more of the steroid nucleus, R⁴ hydrocarbyl group, L⁵hydrocarbyl group or L⁹ hydrocarbyl group.

Heterocyclyloxycarbonyl refers to a heterocyclyloxy group bonded througha carbonyl group to one or more of the steroid nucleus, R4 hydrocarbylgroup, L⁵ hydrocarbyl group or L⁹ hydrocarbyl group.

Heterocyclylcarbonyloxy refers to a heterocyclylcarbonyl group bondedthrough an oxygen atom to one or more of the steroid nucleus, R4hydrocarbyl group, L⁵ hydrocarbyl group or L⁹ hydrocarbyl group.

Alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl and arylcarbonyl refer to moieties wherein acarbonyl group (C═O) provides the carbon atom through which the moietyis joined to one of the steroid nucleus, R4 hydrocarbyl group, L⁵hydrocarbyl group or L⁹ hydrocarbyl group, and an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl or aryl group, respectively, is alsojoined to the carbonyl group.

Alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl and aryloxycarbonyl referto moieties wherein a carbonyl group (C═O) provides the carbon atomthrough which the moiety is joined to one of the steroid nucleus, R4hydrocarbyl group, L⁵ hydrocarbyl group or L⁹ hydrocarbyl group, and analkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy or aryloxygroup, respectively, is also joined to the carbonyl group.

Alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy and aryloxyrefer to groups wherein oxygen is bonded to an alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl or aryl group, respectively, and that oxygen isalso bonded to one of the steroid nucleus, R4 hydrocarbyl group, L⁵hydrocarbyl group or L⁹ hydrocarbyl group.

Alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy and arylcarbonyloxy referto groups wherein oxygen is bonded to an alkylcarbonyl, alkenylcarbonyl,alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl andarylcarbonyl group, respectively, and that oxygen is also bonded to oneof the steroid nucleus, R4 hydrocarbyl group, L⁵ hydrocarbyl group or L⁹hydrocarbyl group.

Alkylsulfide, alkenylsulfide, alkynylsulfide, cycloalkylsulfide,cycloalkenylsulfide and arylsulfide refer to groups wherein sulfur isbonded to an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or arylgroup, respectively, and that sulfur atom is also bonded to one of thesteroid nucleus, R4 hydrocarbyl group, L⁵ hydrocarbyl group or L⁹hydrocarbyl group.

Alkylcarbonylsulfide, alkenylcarbonylsulfide, alkynylcarbonylsulfide,cycloalkylcarbonylsulfide, cycloalkenylcarbonylsulfide andarylcarbonylsulfide refer to groups wherein a sulfur atom is bonded to aalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl or arylcarbonyl group, respectively, and thatsulfur atom is also bonded to one of the steroid nucleus, R4 hydrocarbylgroup, L⁵ hydrocarbyl group or L⁹ hydrocarbyl group.

Alkylene refers to a straight chain bridge of 1 to 5 carbon atoms, whichmay be substituted with 1 to 3 lower alkyl groups or fully or partiallyhalogenated lower alkyl groups.

Alkenylene refers to a straight chain bridge of 2 to 5 carbon atomshaving one or two double bonds, which may be substituted with 1 to 3lower alkyl groups or fully or partially halogenated lower alkyl groups.

Alkynylene refers to a straight chain bridge of 2 to 5 carbon atomshaving one or two triple bonds, which may be substituted with 1 to 3lower alkyl group or fully or partially halogenated lower alkyl groups.

A lower alkyl group refers to C1–C5 alkyl groups, e.g., methyl, ethyl,n-propyl, iso-propyl, n-butyl, t-butyl, sec-butyl, iso-butyl, n-pentyl,iso-pentyl, etc.

Halogen refers to fluorine, chlorine, bromine and iodine, and ahalogenated group refers to a carbon atom having at least one halogenbonded thereto.

Formyl refers to —C(═O)H; hydroxyl refers to —OH; and oxo refers to anoxygen atom which forms part of a carbonyl group.

A pharmaceutically acceptable salt includes acid addition salts and baseaddition salts.

Acid addition salts refer to those salts formed from steroid compoundsof the invention and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

Base addition salts include those salts derived from steroids of theinvention and inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Suitable salts include the ammonium, potassium,sodium, calcium and magnesium salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

In another embodiment, the present invention provides compositions whichinclude a 6,7-dioxygenated steroid compound as described above inadmixture or otherwise in association with one or more inert carriers,as well as optional ingredients if desired. A pharmaceutical compositioncomprising a compound in combination with a pharmaceutically acceptablecarrier or diluent, the compound having the formula

including pharmaceutically acceptable salts and solvates thereof,wherein:

each of C5, C6, C7, C8, C9, C10, C13 and C14 is independentlysubstituted with —X, —R⁴ and —OR¹;

each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted with a substituent selected from (a) or (b), wherein

(a) represents one of: ═O, ═C(R⁴)(R⁴), —C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and—(O(C(R⁴)(R⁴))_(n)O)—, wherein n ranges from 1 to about 6; and

(b) represents two of: —X, —R⁴ and —OR¹, which are independentlyselected at each occurrence;

the A, B, C and D rings may independently be fully saturated, partiallysaturated or fully unsaturated;

R¹ is H or a protecting group such that —OR¹ is a protected hydroxylgroup, where the C6 and C7 —OR¹ groups may together form a cyclicstructure which protects both hydroxyl groups;

R⁴ at each occurrence is independently selected from H and R⁵;

R⁵ is a C₁₋₃₀ organic moiety that may optionally contain at least oneheteroatom selected from the group consisting of boron, halogen,nitrogen, oxygen, silicon and sulfur; where two geminal R⁴ groups maytogether form a ring with the carbon atom to which they are both bonded;and

X represents fluoride, chloride, bromide or iodide;

with the proviso that C15 is not bonded to an oxygen atom.

In preferred compositions: C17 is substituted with a hydrocarbyl group;such as a C₁–C₇ alkyl group; or such as an olefinic group of the formula═C(R⁴)(R⁴), where preferably R⁴ is hydrogen or C₁–C₁₀ alkyl; in apreferred embodiment the C17 hydrocarbyl group excludes—CH(CH₃)(CH₂)₃CH(CH₃)₂. In other preferred compositions, C17 issubstituted with two atoms independently selected from hydrogen andhalogen atoms; or C17 is substituted with at least one oxygen atom; orC17 is substituted with a hydroxyl or protected hydroxyl group; or C17is substituted with a carbonyl or protected carbonyl group; or C17 issubstituted with an alkoxy group. In preferred compositions, thesubstituent at C17 excludes

In other preferred composition, C15 is substituted with two hydrogenatoms; and/or C4 is substituted with hydrogen and one of —X, —R⁵ or—OR¹; and/or C5 is substituted with hydrogen; and/or C4 is bonded to atleast one hydrogen such that when C4 is bonded to two hydrogen atomsthen C3 is not bonded to either oxygen or to two hydrogen atoms. Inother preferred composition, C4 is bonded to two hydrogen atoms onlywhen C3 is not bonded to either oxygen or to two hydrogen atoms. Inanother preferred composition, C4 is bonded to methyl only when C4 isnot bonded to two methyls or formyl. In other preferred compositions,the compounds have a hydrogen at C5 in the alpha configuration. Inanother preferred composition, the compounds have an —OR¹ group at C6with the alpha configuration. In another preferred composition, thecompounds have an —OR¹ group at C7 with the beta configuration. Inanother preferred composition, the compounds have an —OR¹ substituent atC6 with the alpha configuration and an —OR¹ substituent at C7 with thebeta configuration. In other preferred compositions, the compounds haveat least one of C3 and C4 bonded to an oxygen atom, and in a preferredembodiment, both C3 and C4 are bonded to an oxygen atom. In anotherpreferred composition, C10 of the compound is substituted with a methylgroup; and/or C13 of the compound is substituted with a methyl group; orboth C10 and C13 of the compounds are substituted with methyl groups. Ina preferred composition, both C6 and C7 are bonded to hydrogen atoms. Inanother preferred composition, at least one of C1, C2, C3, C4, C5, C8,C9, C10, C11, C12, C13, C14, C15, C16 and C17 is substituted exclusivelywith hydrogen atoms, and more preferably C1 and C2 are substitutedexclusively with hydrogen atoms; and/or C11 and C12 are substitutedexclusively with hydrogen atoms; and/or C15 and C16 are substitutedexclusively with hydrogen atoms. In a preferred composition, thecompounds have a saturated A ring; and/or a saturated B ring; and/or asaturated C ring; and/or a saturated D ring. Compositions with compoundshaving a saturated A ring are preferred, and compositions with compoundshaving fully saturated A, B, C and D rings are more preferred. Inanother preferred composition, the A ring of the compound does notcontain a bicyclic structure. In yet another preferred composition, C3and C4 of the compound are not both substituted solely with hydrogenatoms. These compositions may be used for the treatment of asthma,allergy, inflammation including arthritis, and thrombosis. Thesecompositions may also be formed into a medicament, which may used in thetreatment of, for example, asthma, allergy, inflammation includingarthritis, and thrombosis.

These compositions are useful as, for example, assay standards,convenient means of making bulk shipments, or pharmaceuticalcompositions. An assayable amount of a compound of the invention is anamount which is readily measurable by standard assay procedures andtechniques as are well known and appreciated by those skilled in theart. Assayable amounts of a compound of the invention will generallyvary from about 0.001 wt % to about 80 wt % of the entire weight of thecomposition. Inert carriers include any material which does not degradeor otherwise covalently react with a compound of the invention. Examplesof suitable inert carriers are water; aqueous buffers, such as thosewhich are generally useful in High Performance Liquid Chromatography(HPLC) analysis; organic solvents, such as acetonitrile, ethyl acetate,hexane and the like; and pharmaceutically acceptable carriers.

Thus, the present invention provides a pharmaceutical or veterinarycomposition (hereinafter, simply referred to as a pharmaceuticalcomposition) containing a 6,7-dioxygenated steroid compound as describedabove, in admixture with a pharmaceutically acceptable carrier. Theinvention further provides a pharmaceutical composition containing aneffective amount of a 6,7-dioxygenated steroid compound as describedabove, in association with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be in anyform which allows for the composition to be administered to a patient.For example, the composition may be in the form of a solid, liquid orgas (aerosol). Typical routes of administration include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical composition of the invention areformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a patient take the form of oneor more dosage units, where for example, a tablet may be a single dosageunit, and a container of steroid in aerosol form may hold a plurality ofdosage units.

Materials used in preparing the pharmaceutical compositions should bepharmaceutically pure and non-toxic in the amounts used. It will beevident to those of ordinary skill in the art that the optimal dosage ofthe active ingredient(s) in the pharmaceutical composition will dependon a variety of factors. Relevant factors include, without limitation,the type of subject (e.g., human), the particular form of the activeingredient, the manner of administration and the composition employed.

In general, the pharmaceutical composition includes an active6,7-dioxygenated steroid compounds as described herein, in admixturewith one or more carriers. The carrier(s) may be particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) may begaseous, so as to provide an aerosol composition useful in, e.g.,inhalatory administration.

When intended for oral administration, the composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the composition may beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition willtypically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following adjuvants may be present: binderssuch as carboxymethylcellulose, ethyl cellulose, microcrystallinecellulose, or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin, a flavoring agent such as peppermint,methyl salicylate or orange flavoring, and a coloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it may contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol or a fatty oil.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of the inventive compound suchthat a suitable dosage will be obtained. Typically, this amount is atleast 0.01% of a compound of the invention in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1 and about 70% of the weight of the composition. Preferredoral compositions contain between about 4% and about 50% of the activesteroid compound. Preferred compositions and preparations according tothe present invention are prepared so that a parenteral dosage unitcontains between 0.01 to 1% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the inventive compound of from about 0.1 to about 10%w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The composition may include various materials which modify the physicalform of a solid or liquid dosage unit. For example, the composition mayinclude materials that form a coating shell around the activeingredients. The materials which form the coating shell are typicallyinert, and may be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients may beencased in a gelatin capsule.

The composition in solid or liquid form may include an agent which bindsto the active steroid component(s) and thereby assists in the deliveryof the active components. Suitable agents which may act in this capacityinclude a monoclonal or polyclonal antibody, a protein or a liposome.

The pharmaceutical composition of the present invention may consist ofgaseous dosage units, e.g., it may be in the form of an aerosol. Theterm aerosol is used to denote a variety of systems ranging from thoseof colloidal nature to systems consisting of pressurized packages.Delivery may be by a liquefied or compressed gas or by a suitable pumpsystem which dispenses the active ingredients. Aerosols of compounds ofthe invention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, spacers and the like, which together may form a kit.Preferred aerosols may be determined by one skilled in the art, withoutundue experimentation.

Whether in solid, liquid or gaseous form, the pharmaceutical compositionof the present invention may contain one or more known pharmacologicalagents used in the treatment of asthma, allergy, inflammation (includingarthritis) or thrombosis.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. Various steroid compounds are, and havebeen widely used as active ingredients in pharmaceutical compositionintended for therapeutic use, and accordingly one of ordinary skill inthe art is familiar with preparing such compositions. The steroidcompounds of the present invention may be formulated into pharmaceuticalcompositions in a like manner.

A composition intended to be administered by injection can be preparedby combining the 6,7-dioxygenated steroid with water so as to form asolution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the steroid so as to facilitate dissolutionor homogeneous suspension of the steroid in the aqueous delivery system.

The compounds and compositions described above have utility in treatingallergy and asthma, arthritis and/or thrombosis. The compounds andcomposition described above may also be used to treat a conditionassociated with an elevated level of NFκB, wherein a subject in needthereof is administered an amount of the compound (or compositioncontaining the compound) effective to lower the NFκB activity. As usedherein, “treating allergy and asthma, arthritis and/or thrombosis”refers to both therapy for allergy and asthma, arthritis and thrombosis,and for the prevention of the development of the allergic response,bronchoconstriction, inflammation and the formation of blood clots thatcause thrombosis and associated diseases. As also used herein, NF-kBactivity refers to any increase or decrease in the transcriptionalactivity of genes that is attributable to, directly or indirectly, thebinding of any members of the NF-kB family of proteins to all DNAsequences recognized by this family of proteins.

An effective amount of a compound or composition of the presentinvention is used to treat allergy, asthma, arthritis or thrombosis in awarm-blooded animal, such as a human. Methods of administering effectiveamounts of anti-allergy, anti-asthma, anti-arthritis and anti-thromboticagents are well known in the art and include the administration ofinhalation, oral or parenteral forms. Such dosage forms include, but arenot limited to, parenteral solutions, tablets, capsules, sustainedrelease implants and transdermal delivery systems; or inhalation dosagesystems employing dry powder inhalers or pressurized multi-doseinhalation devices. Generally, oral or intravenous administration ispreferred for the treatment of arthritis and thrombosis, while oral orinhalation/intranasal are preferred for asthma and allergy. The dosageamount and frequency are selected to create an effective level of theagent without harmful effects. It will generally range from a dosage ofabout 0.1 to 100 mg/kg/day, and typically from about 0.1 to 10 mg/Kg/daywhere administered orally or intravenously, for anti-allergy,anti-asthma, anti-arthritis or anti-thrombotic effects. Also, the dosagerange will be typically from about 0.01 to 1 mg/Kg/day whereadministered intranasally or by inhalation for anti-asthma andanti-allergy effects.

Administration of compounds or compositions of the present invention maybe carried out in combination with the administration of other agents.For example, it may be desired to administer a bronchodilator or aglucocorticoid agent for effects on asthma, a glucocorticoid for effectson arthritis, or an anti-histamine for effects on allergy. Non-steroidcompounds may be co-administered with the steroids of the presentinvention, and/or non-steroid compounds may used in combination with thesteroid compounds of the invention to provide a therapy for one or moreof asthma, allergies, arthritis and thrombosis.

The following examples are offered by way of illustration and not by wayof limitation.

Unless otherwise stated, flash chromatography and column chromatographymay be accomplished using Merck silica gel 60 (230–400 mesh). Flashchromatography may be carried out according to the procedure set forthin: “Purification of Laboratory Chemicals”, 3rd. edition,Butterworth-Heinemann Ltd., Oxford (1988), Eds. D. D. Perrin and W. L.F. Armarego, page 23. Column chromatography refers to the processwhereby the flow rate of eluent through a packing material is determinedby gravity. In all cases flash chromatography and radial chromatographymay be used interchangeably. Radial chromatography is performed usingsilica gel on a Chromatotron Model # 7924T (Harrison Research, PaloAlto, Calif.).

A typical work-up procedure for a reaction mixture involves dilution ofthe reaction mixture with an organic solvent (ethyl acetate or diethylether) and washing of the organic mixture with saturated sodiumbicarbonate followed by saturated sodium chloride. The organic layer isthen dried over MgSO₄, the mixture is filtered and the filtrateevaporated to dryness in vacuo to yield the crude product which may ormay not require further purification.

A typical work-up procedure for a Wittig reaction involves firstquenching by the dropwise addition of water. The mixture is then dilutedwith ethyl acetate and washed with saturated sodium bicarbonate and thensodium chloride. The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness.

A typical work-up procedure for a hydroboration reaction involvespouring the reaction mixture into saturated sodium chloride solution(200 ml) followed by extraction of the aqueous slurry with methylenechloride and then washing the combined organic layers with aqueous 25%sodium thiosulphate solution. The organic layer is then dried overmagnesium sulphate, filtered and evaporated to dryness.

Reactions may typically be monitored with thin layer chromatography(TLC) using Silica gel 60 F₂₅₄ plates (EM Science, Gibbstown, N.J.) andan appropriate solvent system. Thin layer chromatography may be carriedout according to the procedure set forth in: “Purification of LaboratoryChemicals”, 3rd. edition, Butterworth-Heinemann Ltd., Oxford (1988),Eds. D. D. Perrin and W. L. F. Armarego, page 30. After elution iscomplete, the TLC plate is dried, lightly sprayed with a 10% solution ofH₂SO₄ in ethanol and then heated until the spots corresponding to thecompounds appear. Unless otherwise stated, filtrations are carried outusing a Whatman (type 1) filter paper.

EXAMPLES Section I Synthesis of 3,4,6,7-polyhydroxylated Steroids

Steroids with the same or closely related ring-structure hydroxylationpattern as compound 237 (shown by the structure below) can besynthesized starting from a number of steroid precursors including4-androsten-3,17-dione (1) and others with C3 oxygen functionalities andΔ⁵ carbon-carbon double bonds such as dehydroisoandrosterone (247).

237 (3β,4α,6α,7β,17β) where R¹═R³═R⁴═H, and R²═R⁵═OH

For example, after protection of the ketone functionalities ofandrosten-3,17-dione (1) (Example 1, Scheme 55) with any one of a numberof appropriate carbonyl protecting groups and concomitant migration ofthe carbon-carbon double bond, allylic oxidation introduces a C7 oxygen.A number of oxidizing agents and experimental conditions can be used forthis oxidation step including but not limited to chromiumtrioxide/3,5-dimethylpyrazole complex, pyridinium chlorochromate (PCC),pyridinium dichromate (PDC), and tBuOOH with RuCl₃. Reduction of theresultant ketone with an appropriate reducing agent gives the hydroxylfunctionality at C7. Several metal hydride reducing agents can be usedfor this task including sodium borohydride and lithium aluminum hydride.Generally, reduction of the C7 carbonyl produces the βOH configurationby hydride attack from the least hindered face of the steroid.

Introduction of the C6 oxygen can be achieved, after protection of theC7 hydroxyl with an appropriate protecting group, by methods such ashydroboration/oxidation or epoxidation followed by ring opening. The Δ⁵carbon-carbon double bond can be epoxidized with any of a number ofperacids including m-chloroperbenzoic acid, trifluoroperacetic acid or3,5-dinitroperoxybenzoic acid. Generally, the epoxide introduced has theα-configuration arising from attack on the least hindered face of thesteroid ring structure. Subsequent ring opening of the epoxide can beaccomplished under acidic conditions such as 80% aqueous acetic acid at60° C. This produces an allylic alcohol at the C6 position with theα-configuration. Alternatively, hydroboration of the Δ⁵ double bond withan appropriate borane complex followed by oxidation using reagents suchas basic hydrogen peroxide will also introduce an hydroxyl group in theα-configuration at C6.

Hydroxyl groups can be introduced at the C3 and C4 positions startingfrom the A-ring 4-ene-3-one functionalization pattern. Reduction of theα,β-unsaturated ketone can be accomplished using lithium dissolved inliquid ammonia. The resultant enolate can be trapped with anelectrophile such as trimethylsilylchloride or diethylchlorophosphate.Hydroboration-oxidation of the silyl enol ether results in theintroduction of an oxygen at C4. This method generates the 3β, 4αhydroxylation pattern. Alternatively, a second reduction using lithiumin liquid ammonia on the enol phosphate produces the Δ³ carbon-carbondouble bond. Epoxidation of the Δ³ double bond with a peracid such asm-chloroperbenzoic acid produces the α-epoxide. Ring opening of thisepoxide could be achieved using a number of acidic or basic conditions.For example, treatment of the 3α, 4α-epoxy functionality with glacialacetic acid in compound 238 (Example 3, Scheme 61) produces the3α-hydroxy, 4β-acetoxy pattern. Removal of the acetate group using anyof a number of reagents including potassium carbonate (or sodiummethoxide) in methanol gives the 3α,4β-hydroxylation pattern.

Example 1 The Steroid 3β,4α,6α,7β,17β-pentahydroxy-5α-androstane (237)can be Synthesized According to the Reaction Sequence Illustrated byScheme 55

Commercially available 4-androsten-3,17-dione (1) (20.0 g, 62.8 mmol) isstirred with ethylene glycol (10 mL) and a catalytic amount ofp-toluenesulfonic acid (1.0 g, 5.2 mmol) in benzene (500 mL) at refluxunder nitrogen for 26 hours (Scheme 56). The water generated by thereaction is removed during this time using a Dean-Stark apparatus. Themixture is then cooled to room temperature and Et₂O is added. Themixture is washed with sodium bicarbonate, then water and dried overmagnesium sulfate. Filtration and concentration gives a pale yellowsolid that is washed with methanol to give the diketal 2 as a whitepowder (14.6 g, 39.0 mmol, 62%). A monoketal byproduct (5.13 g, 15.0mmol) is recovered from the filtrate and recycled. The overall yield,accounting for the byproduct, is 86% of diketal 2.

Allylic oxidation at the C7 position of the diketal 2, using a chromiumtrioxide-3,5-dimethylpyrazole complex in dichloromethane, affordscompound 3 (Scheme 56). Chromium trioxide (46.7 g, 467 mmol) and drydichloromethane (450 mL) are added to a flask under nitrogen, and thencooled to −20° C. using a dry-ice/CaCl₂ solution. 3,5-Dimethylpyrazole(44.9 g, 467 mmol) is added and the mixture is stirred at −20 to −30° C.for 1.5 hours. This is followed by the addition of diketal 2 (7.00 g,18.7 mmol) and continued stirring for 7 hours at −20° C. The reaction isquenched with water and filtered. The filtrate is washed with water, thevolume reduced to 200 mL and then dried over MgSO₄. Filtration andconcentration gives a dark brown oil that is purified using silica gelcolumn chromatography (CH₂Cl₂/EtOAc) to give the enone 3 in 68% yield(4.95 g, 12.8 mmol).

Reduction of enone 3 to the allylic alcohol 4 (Scheme 56) is carried outusing sodium borohydride and cerium (III) chloride heptahydrate in THFand methanol. Freshly distilled THF (200 mL) and methanol (50 mL) areadded to a flask containing the enone 3 (15.3 g, 39.4 mmol) andCeCl₃.7H₂O (16.0 g, 42.9 mmol) under nitrogen. NaBH₄ (3.20 g, 84.6 mmol)is added in portions, and stirring is continued for 1 hour at roomtemperature. Dichloromethane is added, the mixture is washed with NaOH(0.6 N) then water and the organic layer is dried over MgSO₄. Filtrationand concentration yields compound 4 as a white solid (14.1 g, 36.1 mmol,92%).

The C-7 hydroxyl moiety of alcohol 4 is then protected as a silyl ether(Scheme 57). Compound 4 (14.1 g, 36.1 mmol) is dissolved in dry DMF (50mL) then imidazole (5.9 g, 86.7 mmol) and TBDMSCl (6.7 g, 44.5 mmol) areadded, and the mixture is stirred under nitrogen for 5 hours.Dichloromethane is added, the mixture is washed with water, and theorganic layer is dried over MgSO₄. Filtration and concentration gives alight yellow solid that is recrystallized from methanol to give compound5 as a white solid in 61% yield. (11.2 g, 22.2 mmol).

The subsequent epoxidation of compound 5 (Scheme 57) is carried outusing meta-chloroperbenzoic acid (m-CPBA). Compound 5 (0.72 g, 1.4 mmol)is dissolved in dry dichloromethane (5 mL), m-CPBA (0.50 g, 2.9 mmol) isadded and the mixture is stirred vigorously for 20 minutes. Saturatedsodium bicarbonate is added and the aqueous slurry is extracted withdichloromethane. The combined organic extracts are washed sequentiallywith sodium carbonate solution, water, 10% sodium thiosulfate, thenagain with water. Drying (MgSO₄), filtration and concentration gives awhite solid that is purified using flash chromatography to yieldcompound 6 in 77% yield (0.57 g, 1.1 mmol).

Compound 6 is treated with acid to deprotect the C3 and C17 ketones andto open the epoxide moiety to yield the 6-hydroxy-7-silyloxy compound310 (Scheme 57). Aqueous acetic acid (80%, 1 mL) is added to a flaskcontaining compound 6. The mixture is heated at 65° C. for 5 hours,cooled and poured onto dichloromethane. The mixture is washed withsodium bicarbonate and dried over MgSO₄. After filtration andconcentration, the resultant crude product 310 is used in the next stepwithout further purification.

Compound 310 (2.30 mg, 5.32 mmol) in THF (10 mL) is treated withtetrabutylammonium fluoride (TBAF) (8 mL, 1M solution in THF) at roomtemperature under nitrogen for ten minutes in order to remove the silylprotecting group (Scheme 57). The reaction mixture is concentrated andthen purified by flash chromatography (3:1 CH₂Cl₂/EtOAc) to givecompound 7 (1.37 mg, 4.31 mmol) in 81% yield.

Protection of the 6,7-diol of compound 7 is accomplished by treatmentwith 2,2-dimethoxypropane and a catalytic amount of(1S)-(+)-10-camphorsulfonic acid (CSA) to produce acetonide 8 (Scheme58). Compound 7 (1 g, 3.14 mmol) and a catalytic amount of CSA aredissolved in dry DMF (2 mL) and 2,2-dimethoxypropane (10 mL). Themixture is heated at 100° C. for 0.5 hours. Dichloromethane is added andthe mixture is washed with water. The organic layer is dried over MgSO₄,filtered and concentrated to yield compound 8 (1.10 g, 3.07 mmol, 98%)which is used directly in the next reaction without furtherpurification.

Chemoselective reduction of the C-17 carbonyl moiety (Scheme 58),followed by protection of the resultant alcohol as a silyl ether isnecessary. Compound 8 (83 mg, 0.23 mmol) is dissolved in methanol (250mL) under nitrogen and NaBH₄ (15 mg) is added in portions over a periodof 1.5 hours. After an additional 30 minutes, the reaction is quenchedwith acetic acid, then neutralized with NaHCO₃. The methanol isevaporated, and the residue taken up in dichloromethane. The mixture iswashed with water and dried over MgSO₄. Flash chromatography (2:1CH₂Cl₂/EtOAc) gives compound 9 (72 mg, 0.20 mmol, 87%) as the majorproduct.

Protection of the C17 hydroxyl group as a silyl ether to producecompound 10 is followed by reduction of the α,β-unsaturated ketone inthe A-ring using lithium in liquid ammonia-THF, with trapping of theenolate by trimethylsilyl chloride (Scheme 58). A solution of compound10 (75.2 mg, 0.158 mmol) in t-BuOH (0.020 mL) and THF (1.5 mL) istransferred to a flask containing lithium metal in dry, distilledammonia (11.4 mg in 10 mL) at −78° C. After 20 minutes at −78° C.,isoprene (0.5 mL) is added to destroy the excess lithium. The mixture isthen warmed to room temperature and the solvent is evaporated in vacuo.The residue is dissolved in THF (5 mL), cooled to −78° C. and Et₃N (1.1mL, 0.30 mmol) and TMSCl (0.80 mL, 0.30 mmol) are added. The coolingbath is removed and the mixture is stirred for 15 minutes. SaturatedNaHCO₃ is added and this aqueous layer is extracted with Et₂O anddichloromethane. The combined organic layers are washed twice withbrine, dried over MgSO₄ and concentrated. The crude product is purifiedby radial chromatography to give compound 235 in 67% yield (58.5 mg,0.10 mmol).

The C4 hydroxyl is introduced by hydroboration-oxidation of the silylenol ether 235 (Scheme 58). Compound 235 (58.5 mg, 0.106 mmol) isdissolved in dry THF (15 mL) and cooled in an ice-bath. Borane (1.0M THFcomplex: 0.32 mL, 0.32 mmol) is added and the mixture is warmed to roomtemperature and stirred for 45 minutes. More BH₃-THF complex (0.16 mL)is added and stirring is continued for 2 hours. The mixture is thencooled in an ice-bath and 15% NaOH (0.5 mL) and 30% H₂O₂ (0.5 mL) areadded. Vigorous stirring is continued for 2 hours. The aqueous layer isthen extracted with dichloromethane, then Et₂O, and the combined organicextracts are washed with 10% aqueous Na₂S₂O₃, then brine and dried overMgSO₄. The crude product is purified using radial chromatography toyield compound 236 (34.0 mg, 0.0688 mmol, 65%) and the corresponding3β-silyl ether (11.8 mg, 0.0208 mmol, 20%).

Two step deprotection of compound 236 using TBAF in THF then aqueousacidic-THF, gives 3β,4α,6α,7β,17β-pentahydroxy-5α-androstane (237).

Example 2 The Steroid 3α,4α-epoxy-6α,7β,17β-trihydroxy-5α-androstane(239) can be Synthesized According to the Reaction Sequence Shown inScheme 59

3α,4α-Epoxy-6α,7β,17β-trihydroxy-5α-androstane (239) can be producedfrom intermediate 10 in the synthesis of3β,4α,6α,7β,17β-pentahydroxy-5α-androstane (258) (Scheme 60). A solutionof compound 10 (111 mg, 0.234 mmol) in THF (4 mL) is transferred to aflask containing lithium metal in liquid ammonia (6.4 mg in 10 mL) at−78° C. under argon. After 30 minutes at −78° C., isoprene (0.5 mL) isadded to destroy the excess lithium. The mixture is warmed to roomtemperature and the solvent is evaporated in vacuo. The residue isdissolved in THF (5 mL) and cooled to −78° C., then ClP(O)(OEt)₂ (0.044mL, 0.30 mmol) is added and the mixture is stirred for 1 hour. Water anddichloromethane are added, the mixture acidified, and the aqueous layerextracted with dichloromethane and Et₂O. The combined organic layers arewashed with water and dried over MgSO₄. The crude product is purified byradial chromatography to give compound 11 (85.1 mg, 0.139 mmol) in 59%yield.

Reduction of compound 11 with lithium in liquid ammonia in the presenceof t-butyl alcohol produces the olefin 128. A solution of compound 11(85.1 mg, 0.139 mmol) and t-BuOH (0.05 mL) in THF (6 mL) is transferredto a flask containing lithium metal (16 mg) in liquid ammonia at −30° C.under argon. After 30 minutes, the ammonia is allowed to evaporate, thenwater is added. After acidification with aqueous HCl, the mixture isextracted with Et₂O, then EtOAc and the combined organic layers arewashed with water and dried over MgSO₄. The crude product is purified byradial chromatography to give compound 128 (50.3 mg, 0.109 mmol) in 78%yield.

Epoxidation of 128 with meta-chloroperbenzoic acid (m-CPBA) indichloromethane gives the epoxide 238. Compound 128 (50.3 mg, 0.109mmol) is dissolved in dry dichloromethane (1.5 mL) and m-CPBA (43.0 mg)is added. The mixture is stirred at room temperature for 1.5 hours andthen transferred to a separatory funnel and washed with 10% Na₂S₂O₃,saturated NaHCO₃ and water and dried over MgSO₄. Filtration andevaporation of the filtrate gives compound 238 in 78% yield (52.0 mg,0.109 mmol) which is used in the next step without further purification.

Two step deprotection of compound 238 with TBAF in refluxing THF andthen acidic aqueous THF gives compound 239.

Example 3 The Steroid 3α,4β,6α,7β,17β-pentahydroxy-5α-androstane (241)can be Synthesized According to the Following Reaction Sequence (Scheme61)

The synthesis of 3α,4β,6α,7β,17β-pentahydroxy-5α-androstane (241) iscarried out to the intermediate 238 using the same conditions as in thesynthesis of 3α,4α-epoxy-6α,7β,17β-trihydroxy-5α-androstane (239). Thesubsequent epoxide ring opening with concurrent removal of the acetonidefunctionality (Scheme 62) is achieved by heating with glacial aceticacid to yield compound 146, which contains an acetoxy functionality atC4. To a flask containing compound 238 (18.5 mg, 0.038 mmol) is addedacetic acid (0.30 mL). The mixture is stirred with heating at 60° C. for24 hours, then at room temperature for 2 days. The acetic acid isremoved in vacuo to give compound 146 in 93% yield (18 mg, 0.036 mmol).

Deprotection of the C4 hydroxyl using K₂CO₃ in refluxing methanol, anddeprotection of the C17 hydroxyl using TBAF yields the pentahydroxycompound 241.

Example 4 3β,4α,6α,7β,17β-pentahydroxy-5α-androstane 6,7-acetonide (246)

The steroid 3β,4α,6α,7β,17α-pentahydroxy-5α-androstane 6,7-acetonide(246) can be synthesized according to the reaction sequence illustratedby Scheme 63.

Compound 10 is prepared as described in Section 1, Example 1.Deprotection of the 6,7 and 17 hydroxyl moieties is achieved with 80%acetic acid solution and, after appropriate work-up, reprotection ofthese groups as the silyl ether derivative is accomplished to afford242. Compound 10 is dissolved in 80% acetic acid and the mixture isconcentrated in vacuo after stirring for 8 hours at room temperature.The residue is placed in dry DMF containing imidazole and TBDMSCl andstirred for 20 hours at room temperature under a nitrogen atmosphere.Ether is added and the mixture washed with 5% HCl aqueous solution,saturated NaHCO₃ solution and saturated NaCl solution. The organicmixture is dried over MgSO₄, filtered and evaporated. Purification ofthis residue by chromatography over silica gel gives compound 242.

Reduction of the α,β-unsaturated ketone in the A-ring is achieved usinglithium in liquid ammonia-THF, with trapping of the enolate bytrimethylsilyl chloride. A solution of compound 242 in a 1:4 mixture oft-BuOH and THF is transferred to a flask containing lithium metal indry, distilled ammonia at −78° C. After 20 minutes at −78° C., isopreneis added to destroy the excess lithium. The mixture is then warmed toroom temperature and the solvent is evaporated in vacuo. The residue isdissolved in THF, cooled to −78° C. and Et₃N and TMSCl are added. Thecooling bath is removed and the mixture is stirred for 15 minutes.Saturated NaHCO₃ is added and this aqueous layer is extracted with Et₂Oand dichloromethane. The combined organic layers are washed twice withbrine, dried over MgSO₄ and concentrated. The crude product is purifiedby radial chromatography to give compound 243.

The C4 hydroxyl is introduced by hydroboration-oxidation of the silylenol ether 243. Compound 243 is dissolved in dry THF and cooled in anice-bath. Borane (1.0 M THF complex) is added and the mixture is warmedto room temperature and stirred for 45 minutes. More BH₃-THF complex isadded and stirring is continued for 2 hours. The mixture is then cooledin an ice-bath and 30% NaOH and 30% H₂O₂ are added. Vigorous stirring iscontinued for 12 hours. The aqueous layer is then extracted withdichloromethane, then Et₂O, and the combined organic extracts are washedwith 10% aqueous Na₂S₂O₃, then brine and dried over MgSO₄. The crudeproduct is purified using radial chromatography to yield compound 244.

Protection of the 3,4-diol of compound 244 is accomplished by treatmentwith 2,2-dimethoxypropane and a catalytic amount of(1S)-(+)-10-camphorsulfonic acid (CSA) to produce acetonide 245.Compound 244 and a catalytic amount of CSA are dissolved in dry DMF and2,2-dimethoxypropane. The mixture is heated at 100° C. for 0.5 hours.Dichloromethane is added and the mixture is washed with saturated NaHCO₃solution. The organic layer is dried over MgSO₄, filtered andconcentrated to yield compound 245 which is used directly in the nextreaction without further purification.

Compound 245 is then converted to the triol 246 with TBAF. Thus, thetrisilyl ether 245 is dissolved in THF and treated withtetrabutylammonium fluoride (TBAF) (1 M solution in THF) at roomtemperature under nitrogen for 5 hours. The reaction mixture is pouredinto CH₂Cl₂ and washed with brine, dried (MgSO₄) and concentrated invacuo. The residue is then purified by flash chromatography (3:1CH₂Cl₂/EtOAc) to give compound 246.

Section 2 The Synthesis of 22,29-epoxy-15-one Steroids

Steroids that are related to22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165) by thepresence of a C15 ketone and a cyclic hemiacetal functionality in thesteroid side chain (i.e., a C22 hydroxyl functionality condensing with aC29 aldehyde functionality to form a tetrahydropyran ring and a C29hydroxyl group) can be synthesized by a number of methods. The key stepsinclude the introduction of the C15 oxygen, the synthesis of theappropriate side chain (described in Section 3), and the coupling of theside chain. A number of commercially available steroids containingeither a ketone or an acetyl moiety at C17 (e.g., pregnenolone (C17acetyl), 4-androsten-3,17-dione (C17 ketone), dehydroisoandrosterone(C17 ketone) can be used as starting materials.

In one method (Example 5, Scheme 64), the C17 ketone is treated with theWittig reagent prepared by the reaction of ethyltriphenylphosphoniumbromide and potassium t-butoxide in THF in order to introduce a(Z)-17(20) ethylidene moiety. Reaction of the Wittig product with analdehyde-containing compound such as5-acetoxy-3-(1′-methylethyl)-pentanal (156, Section 3, Example 8, Scheme71) in the presence of a Lewis acid gives products that contain therequired C22 oxygen, present as an hydroxyl functionality, and the C29oxygen protected as the acetate. The latter ene reaction gives bothstereoisomers at C22, the ratios of which are dependent on reactionconditions and choice of the aldehyde starting material. Therefore, thefour possible diastereomers arising from the configurations at C22 andC24 can be synthesized by utilizing either the 3R or 3S isomer ofcompound 156.

The ene reaction described above results in a Δ¹⁶ carbon-carbon doublebond which can be utilized to introduce a C15 oxygen via allylicoxidation. For example, after protection of the C22 hydroxyl group ofthe ene product with an appropriate protecting group such ast-butyldimethylsilyl, allylic oxidation, using a reagent such aschromium trioxide/3,5-dimethylpyrazole complex, introduces a ketonefunctionality at the C15 position. Reduction of the Δ¹⁶ double-bondproduces a steroidal compound that has the identical D-ringfunctionality as22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165).

Removal of the C29 protecting group, typically an acetate moiety,followed by oxidation of the resultant primary alcohol produces therequired aldehyde at C29. Removal of the C22 protecting group, generallya t-butyldimethylsilyl group, results in the formation of the side chainhemiacetal moiety found in compound 165.

The second strategy for the attachment of the required hemiacetal sidechain and C15 ketone functionality involves introduction of the C15oxygen before side chain coupling (Example 6, Scheme 67). In this methodthe first step involves conversion of the C17 ketone functionality of asteroidal intermediate to the enol acetate by treatment with isopropenylacetate and p-toluenesulfonic acid. Conversion of the enol acetate tothe α,β-unsaturated ketone is accomplished by treatment with palladiumacetate and tributyltin methoxide.

Introduction of the C15 oxygen is then accomplished using a Michael-typeaddition to the neon by an alkoxide derived from p-methoxybenzyl alcoholin the presence of potassium hydroxide. This functionality can later beselectively deprotected and the resultant alcohol oxidized to the C15ketone.

Elaboration of the C17 ketone functionality to an acetyl group is aprocess that begins with the attack of an acetylide anion. A reagentsuch as commercially available lithium acetylide-ethylene diaminecomplex can be used for this task. Dehydration of the product yields thecompound with a conjugated Δ¹⁶ carbon-carbon double bond. Hydration ofthe acetylene moiety using reagents such as mercury-impregnated Dowexresin in methanol, THF and water produces an acetyl group at C17 (Δ¹⁶,C20 ketone). The Δ¹⁶ carbon-carbon double-bond is reduced using sodiumdithionite and bicarbonate under phase transfer conditions.

The methyl ketone is then converted to an epoxide by treatment with asulphur ylid prepared from trimethylsulphonium iodide and n-butyllithiumin THF. The epoxide is then opened stereoselectively using a Lewis acidsuch as magnesium bromide etherate to give the C22 aldehyde. An alkylanion generated via a lithium-halide exchange reaction from anappropriate halogen, such as the iodide 284 (Example 9, Scheme 72), isthen used to attack the steroidal aldehyde to generate the same sidechain, in a protected form, as found in compound 165. Deprotection ofthe C29 aldehyde produces the desired side chain.

The details of these two strategies are presented in Examples 5 and 6,which follow.

Example 5 22,29-epoxy-3,29-dihydroxy-14β-stigmastan-15-one (260)

As an example of the first method described in the introduction tosection 2, the steroid 22,29-epoxy-3,29-dihydroxy-14β-stigmastan-15-one(260) can be synthesized according to the following reaction sequenceoutlined in Scheme 64.

The synthesis of 22,29-epoxy-3,29-dihydroxy-14β-stigmastan-15-one (260)can be accomplished in ten steps from dehydroisoandrosterone (247).Catalytic hydrogenation of the Δ⁵ carbon-carbon double bond in compound247 yields compound 250, which contains a trans-fused A/B ring-system.Compound 247 is dissolved in EtOAc and 10% Pd/C is added. The mixture isstirred under H₂ at room temperature overnight. Filtration throughcelite and concentration yields compound 250, which can be used directlyin the next reaction.

A Wittig reaction on compound 250 using the phosphorous ylid preparedfrom ethyltriphenylphosphonium bromide and potassium t-butoxide in THFgives compound 251. Ethyltriphenylphosphonium bromide is stirred as asuspension in THF. Potassium t-butoxide is added under a stream ofnitrogen and the mixture stirred at room temperature for 1 hour.Compound 250 is added to the anion thus formed as a solution in THF. Themixture is refluxed for 2 hours under nitrogen then cooled to roomtemperature and quenched by the dropwise addition of water. Saturatedammonium chloride is added and this aqueous layer is extracted withEtOAc and the combined organic phases washed with water and brine anddried over MgSO₄. Filtration and concentration gives the crude product251 which is purified using silica flash chromatography with astep-gradient of hexanes and EtOAc.

Protection of the 3β-hydroxyl is accomplished using t-butyldimethylsilylchloride and imidazole in DMF to yield 252. Compound 251 is dissolved inDMF and imidazole is added. After addition of TBDMSCl, the mixture isstirred overnight at room temperature. Dichloromethane is then added andthe mixture is washed with water and the organic phase dried over MgSO₄.Filtration and concentration yields the crude product 252 which can beused without further purification.

Compound 252 is then coupled with the aldehyde 156 in the presence of anappropriate Lewis acid to yield compound 253. The aldehyde 156 isdissolved in dichloromethane and Me₂AlCl (1.0M in hexane) is added at−78° C. After 5 minutes a solution of compound 252 in dichloromethane isadded. The mixture is then warmed to room temperature over 16 hours. Itis then cooled to −78° C. and quenched with a methanol/water mixture.The layers are separated and the aqueous phase is extracted with Et₂O.The combined organic layers are then washed sequentially with 1N HCl,saturated aqueous NaHCO₃ and brine and then dried over MgSO₄. Afterfiltration and evaporation to dryness, the C22 isomers are separatedusing silica flash chromatography to yield compounds 253a and 253b.

Compound 253a (or 253b) is then dissolved in dry DMF and imidazole isadded. TBDMSCl is then added and the mixture is stirred at roomtemperature for 14 hours and then at 60° C. for 3 hours. The mixture isdiluted with Et₂O, washed with water and then dried over MgSO₄. Afterfiltration and evaporation, the crude product is purified using silicaflash chromatography with EtOAc and hexane mixtures as the eluent togive compound 254.

Allylic oxidation at C15 of compound 254 with CrO₃ and3,5-dimethylpyrazole in dichloromethane gives compound 255. CrO₃ anddichloromethane are added to a flask and cooled to −20° C., and areallowed to stir at this temperature for 15 minutes. 3,5-Dimethylpyrazoleis added and then the reaction stirred for an additional 1.5 hours.Compound 254 in dichloromethane is added and the mixture is kept at −20°C. for 5 days. The mixture is then warmed to room temperature andfiltered through silica gel, washing with EtOAc. Evaporation of thesolvent gives the crude product which is purified using flashchromatography (EtOAc/hexane) to yield pure compound 255.

Reduction of the Δ¹⁶ carbon-carbon double bond in compound 255 can thenbe achieved using hydrogen and palladium on carbon in EtOAc. Compound255 is dissolved in EtOAc and a catalytic amount of 10% Pd/C is added.The mixture is stirred under a hydrogen atmosphere overnight, thenfiltered through Celite and concentrated to yield, after purification,compound 256.

Removal of the acetate protecting group in the side chain of compound256 can then be carried out using potassium carbonate in methanol (orNaOMe in MeOH) to give compound 257. Compound 256 is dissolved inmethanol and K₂CO₃ is added. The mixture is refluxed for 3 hours, cooledto room temperature and poured onto dichloromethane. Aqueous (10%)NaHCO₃ is added and the layers are separated. The aqueous layer isextracted with dichloromethane and the combined organic extracts arewashed with water and dried over MgSO₄. Filtration, evaporation andpurification yields compound 257.

Oxidation of the resultant primary alcohol 257 to the aldehyde 258 canbe achieved using PCC. Compound 257 and NaOAc are stirred indichloromethane and PCC is added. The mixture is stirred at roomtemperature for 3 hours and then filtered through Celite. The filtrateis concentrated and the residue purified using flash chromatography togive compound 258.

Deprotection of both hydroxyl moieties in compound 258 can be achievedin one step using tetrabutylammonium fluoride. Compound 258 is dissolvedin THF and tetrabutylammonium fluoride in THF is added. The mixture isstirred overnight at room temperature and then concentrated in vacuo andpurified to give 22,29-epoxy-3,29-dihydroxy-14α-stigmastan-15-one (248).

Epimerization of compound 248 at the C14 position using KOH in MeOHyields 22,29-epoxy-3,29-dihydroxy-14β-stigmastan-15-one (260). Compound248 is dissolved in MeOH and a solution of KOH in MeOH (25 mg/ml) isadded. The mixture is refluxed for 15 minutes then cooled to roomtemperature. Water is added and the aqueous slurry is extracted withchloroform and then dried over MgSO₄. Filtration and concentration givesthe crude product that contains an epimeric mixture of compounds 248 and260. Separation of 248 and 260 is achieved using column chromatography.

An alternative route to compound 260 (Scheme 65) involves preparation ofthe δ-lactone in the sidechain and subsequent Dibal-H reduction to yieldthe compound containing the C29 hemiacetal functionality. For example,the 3β-hydroxyl in compound 251 is protected as the benzyloxyfunctionality followed by the standard ene reaction described in example5. Deprotection of the C29 acetoxy group is then accomplished usingsodium methoxide in methanol. The resultant diol 263 is then oxidized tothe δ-lactone using silver carbonate on celite in refluxing benzene.Compound 263 is dissolved in benzene and silver carbonate embedded oncelite is added and the mixture refluxed for 12 hours. The reactionmixture is then filtered, evaporated and the residue purified by flashchromatography to yield lactone 264. Allylic oxidation of compound 264using chromium trioxide and 3,5-dimethylpyrazole in dichloromethaneintroduces a carbonyl moiety at C15 (compound 265). Reduction of theconjugated Δ¹⁶ carbon-carbon double bond using hydrogen and palladium oncarbon in EtOAc followed by removal of the benzoate groups using NaOMein 1:1 CHCl₃/MeOH, yields product 266. Finally, protection of the C15ketone as the ethylene ketal followed by selective reduction of theδ-lactone to the lactol is then accomplished using DIBAL at −78° C. anddeprotection using 80% aqueous acetic acid to give compound 260.

Example 6

As stated at the beginning of this section, a second method for theintroduction of the C15 oxygen involves using a Michael addition asdescribed by Cantral et al., J. Org. Chem., 29:64, 1963. Selectiveremoval of the protecting group followed by oxidation of the resultantsecondary alcohol to a ketone at C15 is required. It has been shown byHorita et al., Tetrahedron, 42(11):3021–3028, 1986 that p-methoxybenzylprotecting groups can be removed in the presence of many otherprotecting groups, including benzyl functionalities, using2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). Therefore, the alkoxideproduced from p-methoxybenzyl alcohol in KOH, is used instead of theanalogous benzyloxy alkoxide used by Cantral et al. Scheme 66 belowshows an example of this chemistry on the enone in compound 270, whichis produced in three steps from transdehydroandrosterone (247) usingprocedures described by Takahashi et al., Tetrahedron, 41(24):5747–5754,1985.

The reaction procedures for the Michael Addition illustrated in Scheme67 are as follows. Compound 270 is dissolved in p-methoxybenzyl alcoholand powdered KOH is added. The mixture is stirred under nitrogen at roomtemperature for 4 hours. The mixture is diluted with Et₂O and washedwith water. The organic layer is dried over MgSO₄, filtered andconcentrated. The crude residue is purified using flash chromatographywith a step gradient (EtOAc/hexane) elution to yield compound 271.

Attachment of the sidechain can be accomplished on compounds containinga C17 ketone functionality using a Wittig/ene procedure as illustratedearlier (Example 5), or by using a procedure involving a compoundcontaining a methyl ketone at C17, such as ketone 275. The conversion ofcompounds with C17 ketone functionalities to methyl ketone derivativesis accomplished in a four step process using procedures described in theliterature. An example of this methodology is illustrated in Scheme 68.

The first step in the above process involves conversion of compound 271to the acetylenic alcohol compound 272 using lithiumacetylide-ethylenediamine complex. The acetylide complex is suspended inTHF, and after cooling to −20° C., a solution of compound 271 in THF isadded. The mixture is stirred at room temperature for 6 hours, cooled to0° C. and then quenched with water. The mixture is extracted withdichloromethane then the combined organic extracts are washed with brineand water and then dried over MgSO₄. After filtration and concentrationthe residue is purified using silica flash chromatography (1:6EtOAc/hexane) to yield compound 272.

Dehydration of compound 272 yields the conjugated carbon-carbon doublebond in the D-ring of compound 273. Compound 272 is dissolved in drypyridine and phosphorus oxychloride is added. The mixture is stirred for30 minutes at room temperature and then poured onto ice-water. Theaqueous slurry is extracted with dichloromethane and the combinedorganic extracts are washed with aqueous NaHCO₃ and water and dried overMgSO₄. After filtration and concentration the crude product is purifiedusing silica flash chromatography (1:6 EtOAc/hexane) to yield compound273.

Mercury (2+) impregnated Dowex-50W resin, in a solvent mixture ofmethanol, THF and water is then used to produce the conjugated methylketone 274 from compound 273. Compound 273 is dissolved in methanol/THF(2:1) and two drops of water are added. Hg²⁺/Dowex resin is added andthe mixture is stirred at 60° C. overnight. Filtration and concentrationyields a crude residue which is purified by silica flash chromatography(1:2 EtOAc/hexane) to give methyl ketone 274.

The Δ¹⁶ carbon-carbon double bond in compound 274 is reduced usingsodium dithionite. Compound 274 in toluene is added to a mixture ofsodium dithionite and sodium bicarbonate in water. The phase transfercatalyst Aliquate® 336 is added and the mixture is refluxed for 3 hours.The mixture is extracted with dichloromethane and the organic phase isdried over MgSO₄. Filtration and concentration of the filtrate in vacuogives a crude residue, containing compound 275.

Selective deprotection of the C15 hydroxyl in 275 can be accomplished,at this stage or after coupling of the appropriate sidechain, accordingto the procedures described by Horita et al. (Tetrahedron, 1986, 42(11),3021–3028) which involve oxidation of the p-methoxybenzyl ether with2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in dichloromethane and water.Oxidation of the resultant C15 hydroxyl group to the ketonefunctionality can be accomplished using a number of methods includingPCC in dichloromethane.

Example 7

As described in the introduction to this section, the hemiacetalsidechain functionality in compounds such as 165 can be introduced usingGrignard methodology. This first involves a two step conversion from themethyl ketone functionality at C17 to a C22 aldehyde containingcompound. The methodology for such a conversion, illustrated in Scheme69, has previously been described by a number of groups includingKoreeda et al., Tetrahedron Letters, No. 19, 1641–1644, 1978.

The coupling (Scheme 70) of the desired sidechain to the steroidalaldehyde 278 is achieved by nucleophilic attack of a carbanion. Compound28 is prepared in situ by treatment of the iodide 284 with t-BuLi inEt₂O at −78° C. (see Section 3, Example 9, Scheme 72) and the solutionof the aldehyde 278 in Et₂O is then added. The mixture is stirred for1.5 hours at −78° C. then warmed to room temperature and EtOAc is added.The organic layer is washed sequentially with water, 1N HCl, and water,then dried over MgSO₄. After filtration and concentration, the crudeproduct is purified using silica flash chromatography to yield compound281. Deprotection of the sidechain, using standard procedures such as80% aqueous acetic acid at 60° C., gives the desired sidechainhemiacetal.

Section 3 The Synthesis of Various Side Chain Carbon Skeletons forCoupling to Steroid Nuclei

The synthesis of compound 165, and analogues outlined are convergent(i.e., a highly functionalized side chain is coupled with afunctionalized steroid nucleus). The two methods employed to couple theside chain carbon skeleton to the steroid ring structure were describedin Section 2. Below is a description of the production of the side chaincarbon skeletons to be used in the coupling reactions.

The first method involves the synthesis of5-acetoxy-3-(1′-methylethyl)-pentanal (156) and related aldehydes fromthe primary alcohol 154 (produced from L-carvone, which is availablefrom, e.g., Aldrich Chemical Co., Milwaukee, Wis.). This sequence givesrise to compound 156 with the S-configuration. The compound with theR-configuration is synthesized from D-carvone. It is also noteworthythat any appropriate alcohol protecting group can be used in place ofthe acetate ester to generate an aldehyde compound suitable for couplingto the steroid via an ene-type reaction on compound 252 and relatedsteroids.

The second method (Scheme 72) involves the synthesis of compound 284 andrelated alkyl halides from the carboxylic acid compound 282. Thissequence also generates a product (280) with the S-configuration. Again,the enantiomer of compound 280 is synthesized from D-carvone. As above,any appropriate aldehyde protecting group can be used in place of theketal functionality in 280, for the steps in the production of thecarbanion used in the coupling reaction (generated from a lithium/halideexchange reaction on the corresponding alkyl halide).

Example 8 5-acetoxy-3-(1′-methylethyl)-pentanal (156)

The intermediate 5-acetoxy-3-(1′-methylethyl)-pentanal (156), used inthe Wittig reaction described in previous sections, can be synthesizedaccording to the reaction sequence shown in Scheme 71.

Conversion of L-carvone (153) to compound 154 is accomplished followingliterature procedures (Tetrahedron Letters, 1984, 25(41), 4685–4688).Protection of the primary alcohol in compound 154 is accomplished byconversion to an acetate ester. Compound 154 (207 mg, 1.10 mmol) isdissolved in pyridine (2 mL) and DMAP (10 mg) and acetic anhydride (2mL) are added. The mixture is stirred at room temperature overnight andthen diluted with Et₂O. The mixture is washed with 10% aqueous NaHCO₃then water and dried over MgSO₄. Purification of the crude product isachieved using silica flash chromatography (1:4 EtOAc/hexane) to givecompound 155 (249 mg, 1.08 mmol) in 98% yield.

Removal of the ketal protecting group in ketal 155 using 80% acetic acidgives the desired aldehyde 156. Compound 155 (150 mg, 0.652 mmol) issuspended in 80% AcOH (5 mL) and the mixture is heated at 70° C. for 2hours. The solvent is removed in vacuo and the residue is taken up inEt₂O (50 mL). The mixture is then washed with aqueous NaHCO₃ and brineand then dried over MgSO₄. Filtration and evaporation of the filtrategives pure compound 156 (110 mg, 0.591 mmol) in 91% yield.

Example 9

The intermediate 280, used in the coupling reaction described inprevious sections, can be synthesized according to the reaction sequencesummarized in Scheme 72.

Compound 282 is first synthesized using standard literature procedures(Tetrahedron Letters, 25(41), 4685–4688, 1984) and then converted to 280in a three step procedure. Compound 282 (1.04 g, 5.12 mmol) is dissolvedin dry CCl₄ (120 mL), and I₂ (2.57 g) and iodobenzenediacetate (IDBA)(3.28 g) are added. The mixture is refluxed while irradiating with a 100Watt bulb for 10 minutes. After cooling to room temperature, 5% aqueousNa₂S₂O₃ solution (300 mL) is added until the solution is colorless thenthe layers are separated. The organic phase is washed with water anddried over MgSO₄. Purification gives pure compound 283 (657 mg, 2.30mmol, 45% yield).

Exchange of the aldehyde protecting groups is achieved to producecompound 284 from compound 283 using 2,2-dimethyl-1,3-propanediol andp-toluenesulphonic acid as a catalyst. Compound 283 (42 mg, 0.18 mmol)is dissolved in benzene (5 mL) and 2,2-dimethyl-1,3-propanediol (300 mg)and p-toluenesulphonic acid (3 mg) are added. The mixture is heated atreflux overnight then cooled and evaporated to dryness in vacuo. Theresidue is purified using silica flash chromatography (19:1CH₂Cl₂/Hexanes) to yield compound 284 (40 mg, 0.14 mmol, 80%).

Finally, a lithium iodine exchange reaction on 284 using t-butyllithiumin THF gives the desired product 280. t-BuLi (1.7 M solution in pentane,0.25 mL) is added to a solution of compound 284 (52.1 mg, 0.160 mmol) indry Et₂O (2.0 mL) at −78° C. The solution is stirred at −78° C. until nostarting material remains by GC analysis. This solution is used directlyin the coupling reaction with the aldehyde 278 (Example 7, Scheme 70).

Section 4 The Synthesis of 3,4,6,7-polyhydroxy-22,29-epoxy-15-oneSteroids

The synthesis of polyhydroxylated steroids containing one or both of theC15 ketone and the side chain hemiacetal found in22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165) can beaccomplished using a combination of the methods described in the firsttwo sections. One can either begin with the functionalization of the Aand B rings, then the functionalization of the D-ring and coupling ofthe side chain or the converse. In the compounds containing a C15 ketoneand/or a C29 hemiacetal, the A/B ring can contain 2, 3 or 4 hydroxylgroups at carbons 3, 4, 6 and/or 7 in any combination and in anycombination of configurations.

The following describes synthetic routes to the compounds22,29-epoxy-3,6,7,29-tetrahydroxy-14β-stigmastan-15-one (304) and22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165) and thecorresponding H14α epimers. These are examples of compounds containingthe C15 ketone and the side chain C29 hemiacetal that are derived fromsynthetic transformations described in the first two sections and thesame methodology can be applied in the production of compoundscontaining other hydroxylation patterns at carbons 3, 4, 6 and 7 in theA/B ring system.

Also included below is the synthetic route to22,29-epoxy-3,6,7,29-tetrahydroxystigmastanol (306), a compound thatcontains a polyhydroxylated A/B ring system and a hemiacetal side chainbut lacks the C15 ketone functionality found in compound 165.

Example 10 22,29-epoxy-3,6,7,29-tetrahydroxy-14β-stigmastan-15-one (304)

The steroid 22,29-epoxy-3,6,7,29-tetrahydroxy-140-stigmastan-15-one(304) can be synthesized according to Scheme 73.

The steroid 22,29-epoxy-3,6,7,29-tetrahydroxy-14β-stigmastan-15-one(304) can be synthesized starting from the commercially availablesteroid dehydroisoandrosterone (247). The C17 ketone of compound 247 isfirst protected as the ketal by treatment with ethylene glycol and acatalytic amount of p-toluenesulfonic acid in refluxing benzene.Protection of the 3β hydroxyl as a silyl ether is then achieved usingt-butyldimethylsilyl chloride and imidazole in DMF to give compound 286.Allylic oxidation of compound 286 using chromium trioxide and3,5-dimethylpyrazole introduces a carbonyl moiety at the C7 position(compound 287). Chemoselective 1,2-reduction of this ketone using sodiumborohydride and cerium chloride yields the allylic alcohol 288 in aTHF-methanol solvent system.

Introduction of the C6 alcohol can be accomplished using the sequencedescribed in Example 1, or by hydroboration using a borane-THF complexfollowed by oxidative hydrolysis with basic hydrogen peroxide. In thesecond method, the allylic alcohol in compound 288 is first protected asthe acetate using acetic anhydride and pyridine and the product 289 isdissolved in dry THF and at 0° C. a solution of 1.0 M borane in THF isadded. The mixture is stirred at 0° C. for 30 minutes and then at roomtemperature for 2.5 hours. A 3N NaOH solution is then added dropwisefollowed by 30% aqueous H₂O₂. The mixture is stirred at room temperaturefor 16 hours and then poured into saturated sodium chloride solution.The aqueous slurry is extracted with chloroform and the combined organiclayers are dried over MgSO₄. Filtration and evaporation of the filtrategives a crude product which is purified by flash chromatography to yieldcompound 221.

Protection of the vicinal 6,7-diol 221 is then achieved as thedibenzyloxy derivative using benzyl bromide and sodium hydride indimethylformamide. Compound 221 is dissolved in dimethylformamide andsodium hydride is added. The mixture is stirred for 1 hour at roomtemperature then benzyl bromide is added. Stirring is continued for 2.5hours then the reaction is quenched by the addition of water andstirring is continued for 30 minutes. The mixture is extracted withdiethyl ether and then washed successively with 5% HCl, saturated sodiumbicarbonate and saturated sodium chloride. The organic layer is driedover magnesium sulphate, filtered and evaporated to dryness. Theresultant crude residue is purified using flash chromatography to yieldcompound 291.

Deprotection of the C3 alcohol in compound 291 is achieved using TBAF inTHF at reflux for 2 hours to yield compound 292. Subsequent oxidationusing PDC gives the ketone 293. To affect this transformation, the crudeproduct 292 is dissolved in CH₂Cl₂ and PDC is added. The mixture isstirred at room temperature for 22 hours. Filtration through a bed ofcelite followed by purification of the evaporated filtrate using flashchromatography gives the product ketone 293 as a white solid.

Selective reduction of 293 gives the α-hydroxyl group at C3 in 83%yield. To achieve this, a solution of compound 293 in THF is cooled to−78° C. and LS-Selectride® is added slowly and stirring is continued at−78° C. for 1 hour under nitrogen. Methanol is added and the reactionmixture is warmed to room temperature. Standard work-up followed bypurification by flash chromatography gave compound 294 in approximately80% yield. The 3β-epimer is obtained in approximately 10% yield.Protection of the α-hydroxyl group in compound 294 is achieved as thebenzyloxy derivative. Thus, compound 294 is dissolved indimethylformamide and sodium hydride is added. The mixture is stirredfor 1 hour at room temperature then benzyl bromide is added. Stirring iscontinued for 16 hours then the reaction is quenched by the slowaddition of water and stirring is continued for 30 minutes. The mixtureis extracted with diethyl ether and then washed successively with 5%HCl, saturated sodium bicarbonate and saturated sodium chloride. Theorganic layer is dried over magnesium sulphate, filtered and evaporatedto dryness. The resultant crude residue is purified using flashchromatography to yield compound 295.

Deprotection of the C17 ketal in compound 295 using a mixture of aceticacid, water and acetone (2:1:2) for 14 hours at reflux gives compound296 in 99% yield after purification. Compound 296 is converted to olefin297 using the ylid prepared by the treatment ofethyltriphenylphosphonium bromide with potassium t-butoxide in THF. Thecoupling of the carbon structure of the side chain is achieved using thealdehyde 156 (Scheme 45) and the Lewis acid, dimethylaluminum chloride,in dichloromethane to yield the C22 epimers 298a and 298b afterpurification. Deprotection of the C29 acetoxy group is then accomplishedusing sodium methoxide in methanol. The resultant diol 299 is thenoxidized to the δ-lactone using silver carbonate on celite in refluxingbenzene. Compound 299 is dissolved in benzene and silver carbonateembedded on celite is added and the mixture refluxed for 12 hours. Thereaction mixture is then filtered, evaporated and the residue purifiedby flash chromatography to yield lactone 300. Allylic oxidation ofcompound 300 using chromium trioxide and 3,5-dimethylpyrazole indichloromethane introduces a carbonyl moiety at C15 (compound 301).Reduction of the conjugated Δ¹⁶ carbon-carbon double bond using hydrogenand palladium on carbon in EtOAc gives compound 302. Removal of thebenzoate groups in ester 302 is achieved with concurrent epimerizationat C14 to yield the trihydroxy product 303 which contains the cis C/Dring junction in addition to the trihydroxy product containing the transC/D ring junction which are separable by chromatography. Finally,protection of the C15 ketone as the ethylene ketal ((CH₂OH)₂, pTsOH,benzene) followed by selective reduction of the δ-lactone to the lactolusing DIBAL at −78° C. and deprotection (80% AcOH) can give22,29-epoxy-3,6,7,29-tetrahydroxy-14β-stigmastan-15-one (304).

As an alternative, compound 221 may be treated to remove all of theprotecting group, for example using 80% acetic acid. Thereafter, thehydroxyl groups in the B-ring may be selectively protected. This may bedone with 2,2-dimethoxypropane and camphorsulfonic acid. The ketonegroup at C17 may then be elaborated to an exocyclic double bond usingWittig chemistry, for example, using ethyltriphenylphosphonium bromide,t-BuOK and THF affords ethylidene substitution at C17. Thereafter, theC3 hydroxyl group may be oxidized to a carbonyl group with, e.g., oxalylchloride, DMSO, Et₃N in methylene chloride, followed by reduction of theresulting C3 carbonyl with LS-Selectride® (Aldrich Chemical Co.,Milwaukee, Wis.) to afford the 3-α hydroxy group. Deprotection of thehydroxyl groups in the B-ring then affords compound 330. This is analternative route to compound 330 from what is shown in Scheme 79.

Example 11

Compound 306 can be synthesized from compound 300 according to thereaction sequence in Scheme 74.

Compound 306 can be synthesized from compound 300 in a two step processwith the first step being the deprotection of the hydroxyl groups usinghydrogen and palladium on carbon in EtOAc and EtOH with concurrentreduction of the Δ¹⁶ carbon-carbon double bond. The above mixture isstirred at room temperature for 12 days, filtered and purified by flashchromatography to yield 305. Selective reduction of the δ-lactone to thelactol is then accomplished as follows. Compound 305 is dissolved in THFand cooled to −78° C. DIBAL is added and the mixture is stirred at −78°C. for 3 hours. Standard workup and purification using silica flashchromatography gives give 22,29-epoxy-3,6,7,29-tetrahydroxystigmastanol(306).

Example 12

Reaction conditions described in the previous sections can be applied tothe synthesis of compound 165 as illustrated in Scheme 75.

The synthesis of22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165) iscarried out starting with the commercially available steroid4-androstene-3,17-dione (1). The synthesis from compound 1 to theintermediate 128 has already been described (Example 3, Scheme 61) forthe synthesis of 3α,4β,6α,7β-pentahydroxy-5α-androstanol (241).

Preparing the D-ring of compound 128 for the side chain couplingprocedures described in subsequent examples (Example 16, Scheme 79)begins with the removal of the silyl group at C17 of steroid 128.Compound 128 is dissolved in THF and TBAF (1.0 M in THF) is added. Themixture is heated at reflux for 2 hours and then concentrated in vacuo.The residue is purified by flash chromatography to give alcohol 129.

The hydroxyl moiety in alcohol 129 is convert to the ketone using oxalylchloride in CH₂Cl₂. Compound 129 is dissolved in CH₂Cl₂ and added to asolution of oxalyl chloride in CH₂Cl₂ at −78° C. After stirring at −78°C. for 15 minutes, triethylamine is added and stirring is continued for5 minutes. Standard work-up and purification gave compound 142 as awhite solid.

The conversion of compound 142 to compound 145 is accomplished using thesame procedures described in Example 3 (Scheme 61) for the conversion ofcompound 128 to compound 241. Epoxidation of compound 142 using m-CPBAin dichloromethane to yield epoxide 143 is followed by epoxide openingusing acetic acid to give the 3,6,7-trihydroxy-4-acetoxy compound 144,and removal of the acetate group attached to the oxygen on C4 usingK₂CO₃ in methanol to yield compound 145.

Synthesis of compound 165 from compound 145 is accomplished using thesame types of reactions described in previous sections. Compound 145 isconverted to ethylidene 157 using the ylid prepared fromethyltriphenylphosphonium bromide and potassium t-butoxide in THF. Thefour hydroxyl groups are then protected as benzyl moieties to yield thetetrabenzyloxy compound 158. Coupling of the side chain is achievedusing the aldehyde 156 (Scheme 71) and a Lewis acid such asdimethylaluminum chloride in dichloromethane to yield compound 159.Deprotection of the C29 acetoxy group is then accomplished using sodiummethoxide in methanol. The resultant diol is then oxidized to theδ-lactone 161 using silver carbonate on celite in benzene. Allylicoxidation of compound 161 using chromium trioxide and3,5-dimethylpyrazole in dichloromethane introduces a carbonyl moiety atC15 with concurrent oxidation of the benzyl groups to benzoate groups(compound 162). Reduction of the conjugated Δ¹⁶ carbon-carbon doublebond using hydrogen and palladium on carbon in EtOAc and EtOH givescompound 163. Removal of the benzoate groups in ester 163 is achievedusing basic conditions (for example KOH in MeOH) with concurrentepimerization at C14 to yield product 164 which contains an epimericmixture of the compounds containing the cis C/D ring junction and thetrans C/D ring junction. Finally, protection of the C15 ketone as theethylene ketal ((CH₂OH)₂, pTsOH, benzene) followed by selectivereduction of the δ-lactone to the lactol using DIBAL at −78° C. anddeprotection (80% AcOH) can give22,29-epoxy-3,4,6,7,29-pentahydroxy-14β-stigmastan-15-one (165) and it'sC14 epimer give22,29-epoxy-3,4,6,7,29-pentahydroxy-14α-stigmastan-15-one.

Section 5 Additional Examples of the Synthesis of Novel PolyhydroxylatedSteroids with Biological Activities

In addition to the compounds described in previous sections, a number ofrelated compounds with biological activities have been produced. Theseinclude, among others, compounds containing the 3α,6α,7β-hydroxylationpattern with varying functionalities at the C17 position, as well ascompounds containing the 3β,6α,7β-hydroxylation pattern with varyingfunctionalities at the C17 position. Some of the procedures used for theproduction of these compounds are described in the following examples.

Example 13

A number of 3,6,7-hydroxylated compounds can be prepared from theintermediate compound 221. As described in Scheme 73, compound 221 maybe prepared from the commercially available starting materialdehydroisoandrosterone (247). Specifically, compound 247 (20.0 g, 69.3mmol) is dissolved in benzene (200 ml) in a 500 ml round bottom flaskwhich is then connected to a Dean-Stark apparatus. p-Toluenesulfonicacid monohydrate (0.501 g, 2.64 mmol) is added followed by ethyleneglycol (20 ml) and the mixture is refluxed for 4.5 hours. The mixture iscooled to room temperature and diluted with diethyl ether (200 ml). Theorganic layer is washed with saturated sodium bicarbonate (2×100 ml)then by saturated sodium chloride (2×100 ml). The organic layer is driedwith MgSO₄, filtered and evaporated to dryness to yield the product 285(22.8 g, 68.6 mmol, 99%) which is used in the next reaction withoutfurther purification. Protection of the 3β-hydroxyl in compound 285 as asilyl ether may then be achieved as follows. Compound 285 (22.5 g, 67.7mmol) is dissolved in a mixture of dimethylformamide (112.5 ml) anddichloromethane (112.5 ml). Imidazole (11.3 g, 166.0 mmol) is addedfollowed by t-butyldimethylsilyl chloride (15.8 g, 104.8 mmol). Themixture is stirred at room temperature for 6 hours under Argon thendiluted with diethyl ether (675 ml). The organic mixture is washed withaqueous 5% HCl (2×135 ml) followed by saturated sodium bicarbonate(2×135 ml) then by saturated sodium chloride (2×135 ml). The organiclayer is dried with MgSO₄, filtered and evaporated to dryness to yieldthe crude product 286. The crude product is then recrystallized fromethyl acetate/methanol (3:2) to give compound 286 (25.9 g, 58.0 mmol,83% over two steps) as white crystals. Allylic oxidation of compound 286may then introduce a carbonyl moiety at the C7 position (compound 287).Compound 286 (15.0 g, 33.6 mmol) is dissolved in cyclohexane (60 ml) andH₂O (7.3 ml) is added. Ruthenium (III) chloride hydrate (0.0561 g, 0.27mmol) is added followed by the dropwise addition of t-butylhydroperoxide(37.6 ml). The mixture is stirred at room temperature for 24 hours andthen diluted with ethyl acetate (376 ml). The organic mixture is washedwith saturated sodium chloride (2×188 ml) then with 25% sodiumthiosulphate (2×188 ml). The organic layer is dried with MgSO₄, filteredand evaporated to dryness to yield the crude product. The crude productis recrystallized from ethyl acetate to yield compound 287 (8.6 g, 18.7mmol, 56%). Chemoselective 1,2-reduction of the ketone in compound 287may yield 288. Thus, compound 287 (14.7 g, 31.9 mmol) is dissolved intetrahydrofuran (118 ml) and cerium (III) chloride heptahydrate (17.6 g,47.2 mmol) in methanol (35 ml) is added. The mixture is cooled using anice-bath and sodium borohydride (2.5 g, 66.1 mmol) is added slowly. Themixture is warmed to room temperature and then stirred for 2.5 hours.Aqueous 5% HCl (44 ml) is then slowly added to the mixture followed byethyl acetate (588 ml). The reaction is washed with aqueous 5% HCl (120ml) followed by saturated sodium bicarbonate (120 ml) then by saturatedsodium chloride (120 ml). The organic layer is dried with MgSO₄,filtered and evaporated to dryness to yield the product 288 (14.8 g)which is used in the next reaction without further purification.Compound 288 (14.8 g) is then dissolved in pyridine (30 ml) and aceticanhydride (15 ml) and a catalytic amount of 4-dimethylaminopyridine (30mg) is added. The mixture is stirred at room temperature for 16 hoursand then diluted with ethyl acetate (300 ml). The organic mixture iswashed with saturated sodium bicarbonate (2×60 ml) then by saturatedsodium chloride (2×60 ml). The organic layer is dried with MgSO₄,filtered and evaporated to dryness to yield the crude product.Recrystallization from methanol gives compound 289 (12.6 g, 25.0 mmol,78% yield over two steps). Hydroboration of compound 289 affords the6α,7β-hydroxylation pattern. Thus, compound 289 (8.4 g, 16.6 mmol) isdissolved in dry THF (50 ml) and the mixture is cooled to 0° C. Asolution of 1.0 M borane in THF (20 ml) is added and the mixture isstirred at 0° C. for 30 minutes and then at room temperature for 2.5hours. An aqueous 10N NaOH solution (10 ml) is then added dropwisefollowed by 30% aqueous H₂O₂ (10 ml). The mixture is stirred at roomtemperature for 18 hours and then poured into saturated sodium chloridesolution (200 ml). The aqueous slurry is extracted with methylenechloride (2×250 ml) and the combined organic layers are washed withaqueous 25% sodium thiosulphate solution (2×250 ml) and the organiclayer is dried over MgSO₄. Filtration and evaporation of the filtrategives a crude product which is purified by silica gel flashchromatography (3:1 hexane/ethyl acetate) to yield compound 221 (5.9 g,12.3 mmol, 74%).

Scheme 76 illustrates the synthesis of compounds 326 and 327 fromcompound 221. Compound 221 (1.2 g, 2.4 mmol) is dissolved in aceticanhydride (3 ml) and pyridine (3 ml) and a catalytic amount of4-dimethylaminopyridine (40 mg) is added. The mixture is stirred at roomtemperature for 3 hours then diluted with ethyl acetate (100 ml). Theorganic mixture is washed with aqueous 5% HCl then saturated sodiumbicarbonate (100 ml) and saturated sodium chloride (100 ml). The organiclayer is dried with MgSO₄, filtered and evaporated to dryness to yieldthe product 321 (1.3 g) which is used in the next reaction withoutfurther purification. Removal of the silyl protecting group using TBAFin THF gives compound 322 which contains the 3β-hydroxyl group. Thecrude product 321 is dissolved in THF (10 ml) and 1.0 Mtetrabutylammonium fluoride (4 ml) is added. The mixture is refluxed for1 hour, cooled to room temperature then poured into saturated sodiumchloride solution (50 ml). The aqueous slurry is extracted withmethylene chloride (5×40 ml) and the organic layer is dried over MgSO₄.Filtration and evaporation of the filtrate gives a crude product whichis purified by silica gel flash chromatography (1:1 hexane/ethylacetate) to yield compound 322 (0.85 g, 1.9 mmol, 76% yield over twosteps). Inversion of the stereochemistry at C3 is then accomplished byoxidation using PDC in CH₂Cl₂ to give the ketone 323 followed byL-Selectride® reduction to yield predominantly the 3α-hydroxyl compound324. Thus, compound 322 (0.84 g, 1.9 mmol) is dissolved in CH₂Cl₂ (15ml) and PDC (1.2 g, 3.2 mmol) is added. The mixture is stirred for 40hours at room temperature and then diluted with diethyl ether (50 ml).Filtration and evaporation to dryness gives the crude product which ispurified by silica gel flash chromatography (9:1 hexanes/ethyl acetate)to yield compound 323 (0.81 g, 1.8 mmol, 95%). Compound 323 (0.34 g,0.75 mmol) is then dissolved in THF (10 ml) and then cooled to −78° C.L-Selectride (1.0 M in THF, 1.6 ml) is added and the mixture is stirredat −78° C. for 1 hour. The mixture is warmed to room temperature and anaqueous 10N NaOH solution (1 ml) is then added dropwise followed by 30%aqueous H₂O₂ (1 ml). The mixture is stirred at room temperature for 1hour and then poured into ethyl acetate (50 ml). The organic mixture iswashed with aqueous 5% HCl (2×25 ml) then saturated sodium bicarbonate(2×25 ml) and saturated sodium chloride (2×25 ml). The organic layer isdried with MgSO₄, filtered and evaporated to dryness and purified usingsilica gel flash chromatography (3:1 hexane/ethyl acetate) to yieldcompound 324 (0.214 g, 0.48 mmol, 64%).

Removal of the acetate protecting groups may then be accomplished.Compound 324 (0.25 g, 0.56 mmol) is dissolved in methanol (10 ml) andsodium methoxide (250 mg) is added. The mixture is stirred at roomtemperature for 3 hours and then diluted with ethyl acetate (50 ml). Theorganic mixture is washed with aqueous 5% HCl (2×25 ml) then saturatedsodium bicarbonate (2×25 ml) and saturated sodium chloride (2×25 ml).The organic layer is dried with MgSO₄, filtered and evaporated todryness and purified using silica gel flash chromatography (ethylacetate) to yield compound 325 (0.185 g, 0.51 mmol, 91%). Compound 325(141 mg, 0.385 mmol) is then dissolved in 80% acetic acid (10 ml) andstirred at 70° C. for 14 hours. The mixture is diluted with ethylacetate (50 ml ) and washed with saturated sodium bicarbonate (2×25 ml)and saturated sodium chloride (2×25 ml). The organic layer is dried withMgSO₄, filtered and evaporated to dryness and purified using silica gelflash chromatography (ethyl acetate) to yield compound 326 (0.054 g,0.17 mmol, 44%). Finally, reduction of the ketone 326 (0.023 g, 0.072mmol) by NaBH₄ (0.034 g) in 95% ethanol (1 ml) at room temperature for 2hours produced the tetrahydroxy compound 327 (0.018 g, 0.056 mmol, 78%).

Example 14

As illustrated in Scheme 77, compound 329 can be produced from compound322 in a two step process. Removal of the acetate protecting groups isdone by stirring ester 322 in sodium methoxide and methanol for 15 hoursat room temperature. Deketalization is then accomplished on compound 328using 80% acetic acid to give the trihydroxy compound 329.

Example 15

Compound 329 can also be produced directly from compound 221 in a singlestep using 80% AcOH as shown in Scheme 78. Thus, ketal 221 (1.3 g, 2.7mmol) is dissolved in 80% aqueous acetic acid (20 ml) and the mixture isstirred for 3 hours at room temperature. Evaporation to dryness providescompound 329 (0.79 g, 2.5 mmol, 93%) which is used in subsequentreactions without further purification.

Example 16

The steroid 3α,6α,7β-trihydroxy-17(20)-pregnene (330) can be synthesizedaccording to the reaction sequence shown in Scheme 79.

Compound 330 can be produced from compound 10 (as shown in Scheme 79). Asolution of compound 10 (0.82 g, 1.73 mmol) in diethyl ether (15 ml) istransferred to a flask containing lithium metal (55 mg) in liquidammonia (30 ml) at −78° C. under argon. After 30 minutes at −78° C.,NH₄Cl (2.0 g) is added and the ammonia is evaporated. Water (10 ml) isadded and the layers are separated. The aqueous layer is extracted withCH₂Cl₂ (2×25 ml) and the combined organic layers are washed with water(25 ml) and dried over magnesium sulphate. After filtration andevaporation to dryness, the residue is dissolved in CH₂Cl₂ (15 ml) andPDC (600 mg, 1.59 mmol) is added. The mixture is stirred at roomtemperature for 18 hours then filtered through a bed of celite. Thefiltrate is then purified using silica gel flash chromatography (5:1hexane/ethyl acetate) to give compound 13 (653 mg, 1.40 mmol, 81%).

The ketone 13 is then reduced by the following procedure. Compound 13(1.2 g, 2.53 mmol) is dissolved in THF (30 ml) then the mixture iscooled to −78° C. and L-Selectride® (1.0M in THF, 3.8 ml) is added. Themixture is stirred at −78° C. for 2.5 hours and then warmed to 0° C.Aqueous 10% NaOH (10 ml) is added followed by 30% H₂O₂ (10 ml). Afterstirring for 2 hours, water (20 ml) is added and the aqueous slurry isextracted with CH₂Cl₂ (4×100 ml). The combined organic extracts are thenwashed with 10% Na₂S₂O₃ (2×100 ml) and saturated sodium chloride (2×100ml). The organic layer is dried over magnesium sulphate, filtered andevaporated to dryness to yield the product 14 which is used in the nextstep without further purification. The 3α-hydroxyl group is thenprotected as the acetate using acetic anhydride and pyridine to giveacetate 15. Thus, compound 14 is dissolved in pyridine (15 ml) andacetic anhydride (10 ml) and the mixture is stirred for 12 hours. Ethylacetate and diethyl ether (1:1, 150 ml) are added and the mixture iswashed with 5% HCl (2×50 ml) then by saturated sodium bicarbonate (2×50ml). The organic layer is dried over magnesium sulphate, filtered andevaporated to dryness and the residue is purified by silica gel flashchromatography (10:1 hexane/ethyl acetate) to give compound 15 (0.89 g,1.73 mmol, 68% over two steps). Removal of the silyl protecting group atC17 of compound 15 (0.85 g, 1.63 mmol) is accomplished by refluxing inTHF (30 ml) and TBAF (1.0M in THF, 3.6 ml) for three hours followed byevaporation to dryness. The residue is then dissolved in CH₂Cl₂ (100 ml)and washed with H₂O (3×30 ml). The organic layer is dried over magnesiumsulphate, filtered and evaporated to dryness and the residue is purifiedby silica gel flash chromatography (1:1 hexane/ethyl acetate) to givecompound 16 (0.59 g, 1.45 mmol, 89%). Oxidation of the C17 hydroxyl incompound 16 is then accomplished using oxalyl chloride in DMSO. Thus,compound 16 (0.57 g, 1.40 mmol) is dissolved in CH₂Cl₂ (5 ml) and isadded to a solution of oxalyl chloride (0.15 ml, 1.68 mmol) and DMSO(0.24 ml, 3.36 mmol) in CH₂Cl₂ (10 ml) at −78° C. After stirring at −78°C. for 15 minutes, triethylamine (0.98 ml) is added and stirring iscontinued for 5 minutes. The mixture is warmed to room temperature andH₂O (10 ml) is added. The layers are separated and the organic layer iswashed with 5% HCl (2×5 ml) then by saturated sodium bicarbonate (2×5ml). The organic layer is dried over magnesium sulphate, filtered andevaporated to dryness and the residue is purified by silica gel flashchromatography (2:1 hexane/ethyl acetate) to give compound 17 (0.54 g,1.33 mmol, 95%).

A Wittig reaction on compound 17 using the phosphorous ylid preparedfrom ethyltriphenylphosphonium bromide and potassium t-butoxide in THFgives compound 18. Potassium t-butoxide (0.59 g, 5.23 mmol) is addedunder a stream of nitrogen to a suspension of ethyltriphenylphosphoniumbromide (1.94 g, 5.23 mmol) in THF (15 ml) and the mixture is stirred atroom temperature for 1 hour. Compound 17 (0.53 g, 1.31 mmol) isdissolved in THF (10 ml) and the solution is added to the ylid in THF.The resultant mixture is refluxed for 12 hours under nitrogen thencooled to room temperature. The mixture is filtered through celite andthe filtrate is evaporated to dryness. The residue is dissolved inCH₂Cl₂ (100 ml) and washed with saturated NH₄Cl solution (2×30 ml) andH₂O (2×30 ml). The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness and the residue is purified by silicagel flash chromatography (3:1 hexane/ethyl acetate) to give compound 18(0.33 g, 0.88 mmol, 67%).

Finally, removal of the acetonide group is accomplished. Thus, compound18 (20 mg, 0.053 mmol) is dissolved in 80% aqueous acetic acid (1.5 ml)and stirred at 60° C. for 1 hour. The mixture is evaporated to drynessto yield compound 330 (17.8 mg, 0.053 mmol, 99%).

Example 17

A number of compounds with important biological activities can besynthesized from compound 329. For example, compound 333, which containsthe 3β,6α,7β-hydroxylation pattern and an ethylidene residue at C17, isprepared in three steps from compound 329 (Scheme 80). Thus, compound329 (1.81 g, 5.6 mmol) is dissolved in 2,2-dimethoxypropane (25 ml) anda catalytic amount of camphor sulfonic acid (CSA) (0.03 g) is added andthe mixture is stirred at room temperature for 3 hours. Ethyl acetate(200 ml) is added and the mixture is washed with 5% aqueous HCl (50 ml)then by saturated sodium bicarbonate (2×100 ml) and by saturated sodiumchloride (2×100 ml). The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness and the residue is purified by silicagel flash chromatography (1:1 hexane/ethyl acetate) to give compound 331(1.54 g, 4.3 mmol, 76%). A Wittig reaction on compound 331 using thephosphorous ylid described in previous sections produces compound 332.Thus, potassium t-butoxide (7.15 g, 63.7 mmol) is added under a streamof nitrogen to a stirring solution of ethyltriphenylphosphonium bromide(23.7 g, 63.7 mmol) in toluene (360 ml). The mixture is then stirred for1 hour at room temperature then compound 331 (7.7 g, 21.2 mmol) intoluene (210 ml) is added. The mixture is stirred at room temperaturefor 24 hours under nitrogen then quenched by the dropwise addition ofwater (120 ml). The mixture is diluted with ethyl acetate (900 ml) andwashed with saturated sodium bicarbonate (2×200 ml) sodium chloride(2×200 ml). The organic layer is dried over magnesium sulphate, filteredand evaporated to dryness and the residue is purified by silica gelflash chromatography (2:1 hexane/ethyl acetate) to give compound 332(7.2 g, 19.2 mmol, 90%). Deprotection of the hydroxyl groups in compound332 is then achieved by stirring 332 in 80% acetic acid. Thus, compounds332 (7.2 g, 19.2 mmol) is dissolved in 80% acetic acid (115 ml) and themixture is stirred at room temperature for 3 hours. Evaporation todryness followed by purification by silica gel flash chromatography (9:1CH₂Cl₂/MeOH) gives compound 333 (5.81 g, 17.4 mmol, 90%).

Example 18

Compounds containing a ketone at C3 and a 6,7-hydroxylation pattern canbe obtained from compound 332. For example, oxidation of compound 332using Swern conditions produces compound 334 which can then bedeprotected to compound 335 (Scheme 81). Compound 332 (1.01 g, 2.70mmol) is dissolved in CH₂Cl₂ (10 ml) and then added to a solution ofDMSO (2.5 ml) and 2.0M oxalyl chloride in CH₂Cl₂ (8.1 ml) at −78° C.After stirring at −78° C. for 15 minutes, triethylamine (4.6 ml) isadded and stirring is continued for 15 minutes followed by stirring atroom temperature for 30 minutes. The mixture is diluted with ethylacetate (100 ml) and washed with saturated sodium bicarbonate (2×50 ml)then saturated sodium chloride (2×50 ml). The organic layer is driedover magnesium sulphate, filtered and evaporated to dryness and theresidue is purified by silica gel (pretreated with 1% triethylamine inhexanes) flash chromatography (19:1 hexane/ethyl acetate) to givecompound 334 (0.77 g, 2.07 mmol, 77%). Deprotection of the hydroxylgroups in compound 334 is achieved, as in previous examples, by stirringthe compound 334 (11 mg, 0.030 mmol) in 80% aqueous acetic acid (1.25ml) at room temperature for 1 hour to give, after evaporation to drynessand purification by silica gel flash chromatography (ethyl acetate),compound 335 (9.8 mg, 0.029 mmol, 97%).

Example 19

A by-product of the Swern oxidation in Scheme 81 is the chloroderivative 336. Compound 336 can be deprotected by treatment with 80%aqueous acetic acid as shown in Scheme 82. Thus, compound 336 (0.028 g,0.072 mmol) is dissolved in 80% aqueous acetic acid (2 ml) and stirredat room temperature for 1 hour. The mixture is evaporated to dryness andthe residue is purified by silica gel flash chromatography (3:2hexane/ethyl acetate) to give compound 337 (0.024 g, 0.067 mmol, 94%).

Example 20

As described in an earlier section, compound 330 can be prepared byreduction of the C3 carbonyl in compound 334 (e.g., with LS-Selectride®,Aldrich Chemical Co., Milwaukee, Wis.) to afford the 3α hydroxy groupfollowed by deprotection. Thus, compound 334 (0.85 g, 2.3 mmol) isdissolved in THF (25 ml) and cooled to −78° C. LS-Selectride (1.0M inTHF, 3.0 ml) is added and the mixture is stirred at −78° C. for 3 hours.Aqueous 10N NaOH (2 ml) and 30% H₂O₂ (2 ml) are added and the mixture iswarmed to 0° C. The mixture is diluted with ethyl acetate (150 ml) andwashed with saturated sodium bicarbonate (2×50 ml) then saturated sodiumchloride (2×50 ml). The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness and the residue is purified by silicaflash chromatography (3:1 hexane/ethyl acetate) to give compound 338(0.703 g, 1.88 mmol, 80%). Deprotection of the hydroxyl groups in theB-ring then affords compound 330 (Scheme 83). Thus, compound 338 (1.44g, 3.85 mmol) is dissolved in 80% aqueous acetic acid (25 ml) andstirred at room temperature for 3 hours. The mixture is evaporated todryness to give compound 330 (1.25 g, 3.74 mmol, 97%). This is analternative route to compound 330 from what is shown in Scheme 79.

Example 21

Compounds containing an ethyl residue or another alkyl chain at C17 canbe prepared from the corresponding compound containing an exocyclicdouble at C17. For example, compound 339 is obtained by catalytichydrogenation of the C17–C20 double bond of compound 338, followed bythe deprotection as shown below. Thus, compound 338 (0.15 g, 0.40 mmol)is dissolved 1:1 in acetic acid and ethanol (4 ml) and 10% Pd—C (15 mg)is added. The mixture is stirred under H₂ for 16 hours followed byfiltration and evaporation to yield the desired product 339 (0.115 g,0.340 mmol, 85%) (Scheme 84a). The intermediate 17-ethyl-6,7-acetonide361 is produced if the hydrogenation reaction is carried out in ethylacetate (Scheme 84b). Thus, compound 338 (0.019 g, 0.050 mmol) isdissolved in ethyl acetate (5 ml) and 10% Pd—C (9 mg) is added. Themixture is stirred under H₂ for 14 hours followed by filtration andevaporation to yield the desired product 361 (0.018 g, 0.048 mmol, 96%).

Likewise, compound 332 in acetic acid is hydrogenated using H₂ and 10%Pd—C to yield the product 340 (Scheme 85).

Example 22

Compounds with alternative alkyl groups at C17 can be prepared usingdifferent Wittig reagents. For example, compound 342 is easily obtainedfrom a Wittig reaction, similar to the one described previously butusing MePh₃PBr as the Wittig reagent rather than EtPh₃PBr followed bythe deprotection of triol 341 as shown below (Scheme 86). Thus,methyltriphenylphosphonium bromide (1.97 g, 5.51 mmol) and tBuOK (0.61g, 5.4 mmol) are stirred in THF (10 ml) for 1 hour. Ketone 331 (0.411 g,1.14 mmol) in THF (5 ml) is added to the ylid and the mixture isrefluxed for 3 hours. After standard work-up, as described earlier, thecrude product is purified using silica gel flash chromatography (3:1hexane/ethyl acetate) to give compound 341 (0.332 g, 0.920 mmol, 81%).Product 341 (0.204 g, 0.567 mmol) is then treated with 80% acetic acidsolution (4 ml) for 1.5 hours at room temperature. The mixture isevaporated in vacuo to give the desired triol 342 (0.173 g, 0.540 mmol,95%). The corresponding C17 methyl derivative is then prepared byhydrogenation of compound 341 (0.023 g, 0.063 mmol) and 10% Pd—C (15 mg)in ethyl acetate (5 ml) under H₂ atmosphere followed by, afterfiltration and evaporation to dryness, deprotection with 80% acetic acidsolution (2.5 ml) to afford compound 343 (0.018 g, 0.056 mmol, 88%)(Scheme 87).

Example 23

Compounds containing a methylene carbon at C3 can be synthesized in anumber of different ways. One example involves a modified Bartonprocedure (Robins et al., J. Am. Chem. Soc., 105:4059–4065, 1983) asshown in Scheme 88. The alcohol 332 (0.10 g, 0.27 mmol) is treated withphenyl chlorothionoformate (0.45 ml, 3.3 mmol) in pyridine (2 ml) andmethylene chloride (3 ml) at room temperature for 2 hours. The mixtureis evaporated to dryness and the residue is purified by silica gel flashchromatography (30:1 hexane/ethyl acetate) to give the thionester 344(0.12 g, 0.22 mmol, 84%). The ester 334 (0.091 g, 0.17 mmol) is thentreated with nBu₃SnH (60 μl, 0.22 mmol) and a catalytic amount of AIBN(4 mg) in toluene (3 ml) at 75° C. for 3 hours under an inertatmosphere. The mixture is evaporated to dryness and compound 345 (0.035g, 0.1 mmol, 57%) is obtained upon purification using silica gel flashchromatography (30:1 hexane/ethyl acetate). Treatment of compound 345(0.025 g, 0.069 mmol) with 80% aqueous acetic acid (2 ml) for 1 hour atroom temperature followed by evaporation to dryness and purification bysilica gel flash chromatography (3:1 hexane/ethyl acetate) gives thediol 346 (0.021 g, 0.066 mmol, 96%).

Example 24

Compounds containing a methylene carbon at C17 can be produced usingsimilar chemistry to that described in Example 23. For example, thealcohol group of compound 331 (0.15 g, 0.42 mmol) is initially protectedas a silyl ether by treatment with t-butyldimethylsilyl chloride (0.095g, 0.63 mmol) and imidazole (0.057 g, 0.84 mmol) in dimethylformamide (4ml) at room temperature for 4 hours. The mixture is diluted with diethylether (75 ml) and washed with saturated sodium bicarbonate (2×25 ml)then H₂O (2×25 ml). The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness and the residue is purified by silicaflash chromatography (9:1 hexane/ethyl acetate) to give compound 347(0.18 g, 0.38 mmol, 90%) (Scheme 89). The ketone function of compound347 is then reduced with lithium aluminum hydride in diethyl ether toafford the 17β-alcohol 348. Thus, compound 347 (0.18 g, 0.37 mmol) isdissolved in diethyl ether (5 ml), cooled to 0° C. and lithium aluminumhydride (0.018 g, 0.48 mmol) is added. The mixture is stirred at 0° C.for 30 minutes then saturated sodium bicarbonate (1 ml) is addeddropwise. The mixture is diluted with diethyl ether (50 ml) and washedwith saturated sodium bicarbonate (2×15 ml) then by H₂O (2×15 ml). Theorganic layer is dried over magnesium sulphate, filtered and evaporatedto dryness and the residue is purified by silica flash chromatography(4:1 hexane/ethyl acetate) to give compound 348 (0.15 g, 0.31 mmol,85%). Using a Barton type reaction, compound 348 is treated with NaH,CS₂ and MeI in THF to produce the methyl xanthate 349 after work-up andpurification. Thus, compound 348 (0.052 g, 0.11 mmol) is dissolved inTHF (5 ml) and NaH (17.4 mg (60% in oil), 0.43 mmol) and imidazole (5mg, 0.074 mmol) are added. The mixture is stirred at room temperaturefor 30 minutes then carbon disulfide (0.2 ml) is added and stirring iscontinued for 2 hours followed by refluxing for 30 minutes. MeI (0.2 ml)is added and refluxing is continued for an addition 30 minutes. H₂O (1ml) is added dropwise and the mixture is diluted with diethyl ether (100ml) and washed with 5% HCl (2×30 ml) then saturated sodium bicarbonate(2×30 ml) and then H₂O (2×30 ml). The organic layer is dried overmagnesium sulphate, filtered and evaporated to dryness and the residueis purified by silica gel flash chromatography (15:1 hexane/ethylacetate) to give compound 349 (0.054 g, 0.09 mmol, 85%). In the nextreaction AIBN is typically used as the radical initiator. Thus, compound349 (0.05 g, 0.087 mmol) is dissolved in toluene (15 ml) and nBu₃SnH(0.051 g, 0.17 mmol) and a catalytic amount of AIBN (10 mg) are addedand the mixture is refluxed for 22 hours under an inert atmosphere. Themixture is cooled to room temperature, evaporated to dryness andpurified using silica flash chromatography (40:1 hexane/ethyl acetate)to give compound 350 (0.010 g, 0.022 mmol, 25%). Treatment of compound350 (0.010 g, 0.022 mmol) with 80% acetic acid (2 ml) for 18 hours atroom temperature followed by evaporation to dryness and purificationusing silica gel flash chromatography (20:1 CHCl₃/MeOH) gives compound351 (0.0065 g, 0.021 mmol, 96%).

Example 25

Compounds with higher alkyl chains attached to C17 can be produced usingsimilar chemistry to that described in previous examples. For example,compound 354 can be produced in 4 steps from commercially availablecholesteryl acetate (228), as shown in Scheme 90. Methodology previouslydescribed involving C7 oxidation using RuCl₃ and tBuOOH followed byreduction of the C7 ketone using NaBH₄/CeCl₃ affords alcohol 352.Acetylation of compound 352 followed by hydroboration (and subsequentalkaline-peroxide workup) produces the desired compound 354.

Specifically, compound 228 (0.431 g, 1.01 mmol), RuCl₃ (9.020 g, 0.098mmol), cyclohexane (5 mL), water (0.25 mL) and 70% tBuOOH in water (1.5mL, 11.0 mmol) are stirred for 24 hours at room temperature. The mixtureis diluted with ethyl acetate (125 ml) and washed with an aqueoussolution of 10% Na₂SO₃ (2×50 ml) and saturated NaCl (2×50 ml). Theorganic layer is dried with MgSO₄ and evaporated to dryness.Purification by silica gel flash chromatography with 9:1 hexanes-ethylacetate affords compound 229 (0.263 g, 0.591 mmol, 59%). The reductionof the C7 ketone proceeds as follows. A mixture of CeCl₃7H₂O (2.00 g,5.368 mmol) in methanol (5 ml) is added to a solution of ketone 229(1.11 g, 2.52 mmol) in THF (5 ml) and the mixture is cooled to 0° C.NABH₄ (0.119 g, 5.14 mmol) is added and the mixture stirred at 0° C. for1 hour followed by warming to room temperature and continued stirringfor 2 hours. The mixture is cautiously quenched with aqueous 5% HCl (10ml) and diluted with ethyl acetate (250 ml). The emulsion is then washedwith aqueous 5% HCl (2×100 ml), saturated aqueous NaHCO₃ (2×100 ml), andsaturated aqueous NaCl (2×100 ml). The organic phase is dried with MgSO₄and evaporated to dryness. The residue is purified by silica gel flashchromatography with 9:1 hexanes-ethyl acetate giving alcohol 352 (0.850g, 1.91 mmol, 76%). Protection and hydroboration are then accomplished.Thus, compound 352 (0.850 g, 1.91 mmol), pyridine (5 ml) and aceticanhydride (5 mL) are stirred at room temperature for 16 hours. Themixture is diluted with ethyl acetate (150 ml) and washed with aqueous5% HCl (3×50 ml), saturated aqueous NaHCO₃ (2×50 ml), and saturatedaqueous NaCl (2×50 ml). The organic phase is dried with MgSO₄ andevaporated to dryness. The residue is purified by silica gel flashchromatography with 19:1 hexanes-ethyl acetate giving product 353 (0.823g, 1.70 mmol, 99%). The diacetate 353 (0.275 g, 0.5648 mmol) in THF (5ml) is cooled to 0° C. and BH₃ in THF (1.0 M, 2.5 mL, 2.5 mmol) isadded. The mixture is stirred for 3 hours at 0° C., then cautiouslyquenched with an aqueous 10 N NaOH solution (1 mL) and an aqueous 30%H₂O₂ (1 ml) solution. The resultant mixture is stirred for 16 hours,diluted with ethyl acetate (100 ml) and washed with an aqueous 10%Na₂SO₃ solution (2×50 ml), a saturated aqueous NaHCO₃ solution (2×50 ml)and saturated NaCl solution (2×50 ml). The organic phase is dried overMgSO₄ and evaporated to dryness. Purification by silica gel flashchromatography (3:1 hexanes-ethyl acetate) affords the product3β-acetoxy-6α,7β-dihydroxy-5α-cholestane (0.032 g, 0.069 mmol, 13%)which is deprotected by treatment with sodium methoxide (prepared fromsodium metal (0.262 g, 11.4 mmol) and methanol (10 ml)) at roomtemperature for 1.5 hours. The mixture is diluted with ethyl acetate (30ml) and then washed with a saturated aqueous NaHCO₃ solution (2×15 ml)and saturated aqueous NaCl solution (2×15 ml). The organic layer isdried with MgSO₄ and evaporated to dryness. The residue is purified bysilica gel flash chromatography with 1:1 hexanes-ethyl acetate givingtriol 354 (0.029 g, 0.069 mmol, 99%).

Example 26

Compounds containing additional functional groups in the A-ring havebeen prepared and tested for biological activity. For example, compound360 can be prepared in a multi-step synthesis from compound 335, asillustrated in Scheme 91. Acetylation of compound 335 using aceticanhydride and pyridine and DMAP to give the diacetoxy compound 355.Thus, compound 335 (1.5 g, 4.5 mmol) pyridine (10 ml), acetic anhydride(5 mL) and 4-dimethylaminopyridine (0.028 g, 0.23 mmol) are stirred atroom temperature for 12 hours. The mixture is diluted with ethyl acetate(300 ml) and washed with aqueous 5% HCl (3×50 ml), saturated aqueousNaHCO₃ (3×50 ml), and H₂O (3×50 ml). The organic phase is dried withMgSO₄ and evaporated to dryness to yield a crude residue 355 (1.9 g)which is used in the next reaction without further purification. Thus,crude product 355(0.800 g, 1.9 mmol) is dissolved in AcOH and 10% Pd—C(80 mg) is added. The mixture is then stirred under H₂ atmosphere for 16hours at room temperature. The mixture is filtered and evaporated todryness to yield a crude residue which is purified by silica gel flashchromatography (5:1 hexane/ethyl acetate) to give compound 356 (0.702 g,1.67 mmol, 88%). The oxime 357 is then obtained by refluxing compound356 with HONH₂—HCl in a MeOH-pyridine solution. Thus, compound 356 (0.05g, 0.12 mmol) is dissolved in a mixture of pyridine (2 ml) and methanol(2 ml) and HONH₂—HCl (0.017 g, 0.24 mmol) is added. The mixture isrefluxed for 1.5 hours and the mixture is diluted with ethyl acetate (50ml) and washed with 5% HCl (3×15 ml) then by saturated sodiumbicarbonate (3×15 ml) and then H₂O (3×15 ml). The organic layer is driedover magnesium sulphate, filtered and evaporated to dryness to yieldcompound 357 (0.052 g, 0.12 mmol, 99%) which is used in the nextreaction without further purification. Product 357 (0.071 g, 0.16 mmol)is then dissolved in pyridine (13 mg) in acetic anhydride (3 ml), cooledto 0° C. and acetyl chloride (15.5 mg, 0.20 mmol) is added. The mixtureis then heated for 8 hours at 100° C. H₂O (0.5 ml) is added and heatingis continued for 30 minutes. The mixture is then cooled to roomtemperature, diluted with H₂O (10 ml), and extracted with CH₂Cl₂ (3×15ml). The combined organic extracts are then washed with H₂O (2×10 ml).The organic layer is dried over magnesium sulphate, filtered andevaporated to dryness and the residue is purified by silica gel flashchromatography (2:1 hexane/ethyl acetate) to give compound 358 (0.41 g,0.086 mmol, 52%). The C3 ketone in 358 (0.020 g, 0.042 mmol) is thenreduced with NaBH₄ (2.4 mg) in THF (2 ml) at room temperature for 1hour. AcOH (2 drops) is added and the mixture is diluted with ethylacetate (50 ml) and washed with saturated sodium bicarbonate (2×15 ml)then H₂O (2×50 ml). The organic layer is dried over magnesium sulphate,filtered and evaporated to dryness and the crude product 359 isdissolved in methanol (1.5 ml). NaOMe (10 mg) is added and the mixtureis stirred at room temperature for 48 hours. Amberlite IR-120 ionexchange resin is added until pH 6. The mixture is filtered andevaporated to dryness and purified by silica gel flash chromatography(10:1 CHCl₃/MeOH) to give compound 360 (0.010 g, 0.028 mmol, 67% overtwo steps).

The following examples are offered by way of illustration and not by wayof limitation.

Utility Examples

The compounds described above have utility in treating allergy andasthma, arthritis and/or thrombosis. As used herein, “treating allergyand asthma, arthritis and/or thrombosis” refers to both therapy forallergy and asthma, arthritis and thrombosis, and for the prevention ofthe development of the allergic response, bronchoconstriction,inflammation and the formation of blood clots that cause thrombosis andassociated diseases. An effective amount of a compound or composition ofthe present invention is used to treat allergy, asthma, arthritis orthrombosis in a warm-blooded animal, such as a human. Methods ofadministering effective amounts of anti-allergy, anti-asthma,anti-arthritis and anti-thrombotic agents are well known in the art andinclude the administration of inhalation, oral or parenteral forms. Suchdosage forms include, but are not limited to, parenteral solutions,tablets, capsules, sustained release implants and transdermal deliverysystems; or inhalation dosage systems employing dry powder inhalers orpressurized multi-dose inhalation devices. Generally, oral orintravenous administration is preferred for the treatment of arthritisand thrombosis, while oral or inhalation/intranasal are preferred forasthma and allergy. The dosage amount and frequency are selected tocreate an effective level of the agent without harmful effects. It willgenerally range from a dosage of about 0.01 to 100 mg/kg/day, andtypically from about 0.1 to 10 mg/Kg/day where administered orally orintravenously, for anti-allergy, anti-asthma, anti-arthritis oranti-thrombotic effects. Also, the dosage range will be typically fromabout 0.01 to 1 mg/Kg/day where administered intranasally or byinhalation for anti-asthma and anti-allergy effects.

Administration of compounds or compositions of the present invention maybe carried out in combination with the administration of other agents.For example, it may be desired to administer a bronchodilator or aglucocorticoid agent for effects on asthma, a glucocorticoid for effectson arthritis, or an anti-histamine for effects on allergy. Non-steroidcompounds may be co-administered with the steroids of the presentinvention, and/or non-steroid compounds may used in combination with thesteroid compounds of the invention to provide a therapy for one or moreof asthma, allergies, arthritis and thrombosis.

For example, provided below are several examples of the biologicalactivity of various compounds described in Synthesis Examples, Sections1–5.

Anti-Thrombolytic Activity of Polyhydroxylated Steroids

Within the present invention, it was discovered that thepolyhydroxylated steroids as well as intermediates described in previoussections inhibited the aggregation of platelets caused by plateletactivating factor (PAF). PAF is a local mediator of thrombosis andprevention of the formation of blood clots has direct implication in thetreatment of thrombosis and associated cardiovascular diseases. Theassay system used to evaluate the ability of compounds to inhibit theaggregation of platelets in response to exogenous stimuli is indicativeof anti-thrombotic or thrombolytic activity.

Platelets were isolated from rabbit blood and prepared at a density of2.4×10⁸ cells/ml in Tyrodes buffer (pH 7.2) containing Ca²⁺. Plateletswere incubated with each compound for 5 min at 37° C. prior tostimulation. Platelets were stimulated with 1 nM platelet activatingfactor (PAF; EC₇₅) in the presence of each of the compounds andaggregation was monitored for 5 min. Compounds were solubilized indimethylsulfoxide (DMSO) and aggregation was measured as a percentage ofthe response to 1 nM PAF obtained in the presence of the appropriateconcentration of DMSO. The degree of inhibition caused by each samplewas calculated using the control response in the presence of DMSO equalto 100%.

Table 1 shows examples of some of the compounds that inhibit plateletaggregation in response to PAF.

TABLE 1 THE EFFECT OF VARIOUS COMPOUNDS ON THE AGGREGATION OF RABBITPLATELETS STIMULATED WITH 0.1 NM PAF* Sample % Inhibition Number at 80μM 7 12.3 8 11.6 165 51.2 236 100 241 93.1 246 21 330 20.9 *Plateletswere incubated with 80 μM of each compound for 5 minutes prior tostimulation. Responses were measured as a percentage of the inhibitionof the PAF-induced response obtained in the presence of the appropriateconcentration of DMSO alone.Effects of Compounds on the Release of Hexosaminidase from a Rat MastCell Line (RBL-2H3)

The anti-allergic effects of various polyhydroxylated steroids of thepresent invention were evaluated by measuring their effect onantigen-induced secretion of hexosaminidase from a passively sensitizedrat mast cell line (RBL-2H3) and a murine mast cell line (MC/9). Theability of agents to inhibit the release of mast cell granule contents,e.g., histamine and hexosaminidase, is indicative of anti-allergy and/oranti-asthma activity.

Hexosaminidase is released from the mast cell granule along withhistamine and other mediators during antigen challenge. RBL-2H3 and MC/9cells were grown in culture and passively sensitized to dinitrophenol(DNP) using anti-human-DNP (IgE). Cells were incubated with eachcompound (25 μM) for 1 hour at 37° C. and then stimulated with 0.1 mg/mlDNP-HSA (antigen) for 15 min. Aliquots of the supernatant were removedand used to measure the amount of hexosaminidase released duringchallenge with the antigen. The amount of hexosaminidase present in thesupernatant was determined calorimetrically by monitoring the enzymaticmetabolism of p-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG) over aperiod of 1 hour at 410 nm. The effect of each compound was determinedas a percentage of the antigen-induced response (minus backgroundrelease) obtained in the presence of DMSO alone, as set forth in Tables2 and 3. These values were used to determine the degrees of inhibitionof antigen-induced hexosaminidase release from the cells.

TABLE 2 THE EFFECT OF VARIOUS COMPOUNDS (EACH 25 μM) ON ANTIGEN-INDUCEDRELEASE OF HEXOSAMINIDASE FROM PASSIVELY SENSITIZED RAT MAST CELLS(RBL-2H3) AND MOUSE MAST CELLS (MC/9)* Percentage Inhibition (mean)Compound Number RBL-2H3 Cells MC/9 cells 333 69 71 335 56 69 339 79 67337 44 56 339 76 55 330 59 49 343 52 40 342 57 39 361 ND 13 331 ND 5 35430 76 346 16 61 ND = not determined.

TABLE 3 THE EFFECT OF VARIOUS COMPOUNDS (EACH 25 μM) ON ANTIGEN-INDUCEDRELEASE OF HEXOSAMINIDASE FROM PASSIVELY SENSITIZED RAT MAST CELLS(RBL-2H3)* Percentage Inhibition (mean) RBL-2H3 Cells 7 21.7 165 51.8236 31 241 22 246 29.4 306 40.5 322 25.6 327 35.2 328 34.3 266 −12.5 2399.6 351 33.6 360 76.0 *Values represent the percentage inhibitionproduced by each compound compared to the response obtained in thepresence of DMSO alone.Effects of Selected Compounds on Allergen-Induced Contraction of IleumSmooth Muscle

The ability of compounds to inhibit allergen-induced contraction ofileum from sensitized animals is indicative of antiallergic activity.Sensitized guinea pig ileum is particularly useful in measuring theimmediate allergic response.

The guinea pig ileum has been used to evaluate the ability of compoundsto inhibit allergen-induced histamine and mediator release causingsmooth muscle contraction. Guinea pigs were sensitized by anintraperitoneal injection of 100 mg ovalbumin and an intramuscularinjection of 50 mg ovalbumin on day 0, followed by a secondintramuscular injection of 50 mg ovalbumin on day 1. Twenty one daysafter the initial immunization, the animals were found to be sensitized,in that an anaphylactic response was obtained upon challenge withallergen. Segments of ileum were prepared and suspended, with musclecontractions being measured in the longitudinal plane, in Tyrode'sbuffer at 37° C. and aerated with 5% CO₂ in O₂. Tissues were suspendedunder a resting tension of 2 grains and isometric contractions weremeasured using force-displacement transducers coupled to a polygraph.Tissues were stimulated with 3 μM histamine 3 times to ensurereproducible contractions were obtained. Tissues were then incubatedwith each compound (30 μM) or 0.15% dimethylsulphoxide (DMSO) as acontrol, for 20 min, after which time the tissues were challenged with100 μg/ml ovalbumin. The magnitude of the contraction induced by OA inthe presence of each compound was expressed as a percentage of thecontraction obtained to 3 μM histamine. The protective effects of thevarious compounds on OA-induced contraction of guinea pig ileum fromsensitized animals are summarized in Table 4.

TABLE 4 THE EFFECT OF VARIOUS COMPOUNDS (EACH 30 μM) ON ANTIGEN-INDUCEDCONTRACTION OF ILEUM FROM SENSITIZED GUINEA PIGS. ANTIGEN-INDUCEDCONTRACTIONS WERE EXPRESSED AS A PERCENTAGE OF THE CONTRACTION INDUCEDBY 3 μM HISTAMINE*. Compound Number Percentage Inhibition 330 70.0 22160.7 338 64.0 7 54.0 333 63.0 343 48.9 336 79.3 342 28.3 339 40.6 33530.7 337 50.7 165 27.0 251 −10.5  (stim) 361 55.0 331 14.5 339 36.3 34662.5 334 74.0 266 17.4 351 43.6 360 50.5 *Values represent the meanpercentage inhibition produced by each compound compared to the responseobtained in the presence of DMSO alone, n = 3–4.Effects of Selected Compounds on Allergen-Induced Bronchoconstriction InVitro and In Vivo

The effects of a number of the compounds described herein onallergen-induced bronchoconstriction were evaluated for anti-asthmaactivity. The ability of a compound to inhibit allergen-induceddecreases in lung function in sensitized guinea pigs in response toantigen-challenge is indicative of anti-asthma activity. In particular,the model system is useful, in the evaluation of the potential effectsof a compound in the treatment of the early asthmatic reaction (EAR)when severe bronchoconstriction occurs.

Guinea pigs were exposed to a nebulized solution of 1% ovalbumin (OA) insaline for 15 min. After 10 days the animals were found to besensitized, i.e., the tracheal tissue responded with anaphylacticbronchospasm to further antigen (OA) challenge. Trachea from theseanimals were found to respond in a similar manner to the in vivosituation. Tracheal rings were prepared and bathed in Krebs-Henseleitsolution at 37° C. and aerated with 5% CO₂ in O₂. Tissues were suspendedunder a resting tension of 2 g and isometric contractions were measuredusing force-displacement transducers coupled to a polygraph. Tissueswere incubated with each compound or 0.1% dimethylsulfoxide (as acontrol) for 20 min, after which increasing concentrations of OA(0.001–100 μg/ml) were added to the tissue. After the finalconcentration of OA was added and the response was recorded, the tissueswere stimulated with 100 μM methacholine which caused maximumcontraction of the trachea. The magnitude of the contraction induced byOA in the presence of each compound was expressed as a percentage of themaximum contraction obtained using methacholine (100 μM). The protectiveeffects of various compounds on OA-induced contraction of trachealtissue are summarized in Tables 5–7.

TABLE 5 EFFECTS OF SELECTED COMPOUNDS (EACH 20 μM) ON ALLERGEN-INDUCEDCONTRACTIONS OF ISOLATED TRACHEA* (STUDY 1) μg/ml OA 0.001 0.003 0.010.03 0.1 0.3 1.0 3.0 10.0 30.0 Ctrl 2.9 7.7 11.4 17.4 19.7 26.4 30.937.2 43.6 46.8 241 8.5 12.6 17.1 25.8 31.0 33.4 36.3 34.4 35.0 37.0 2361.9 5.6 5.6 5.6 11.1 16.7 39 28 39 41 145 0.8 6.2 5.9 7.6 8.3 13.6 14.820.0 28.0 31.0 246 2.6 4.0 6.5 9.7 12.2 15.3 20.0 21.0 23.0 22.0 *Valuesrepresent percentage contraction compared to that obtained with 100 μMmethacholine (100%), Ctrl = control (0.1% DMSO)

TABLE 6 EFFECTS OF VARIOUS COMPOUNDS (EACH 20 μM) ON ALLERGEN-INDUCEDCONTRACTIONS OF ISOLATED TRACHEA* (STUDY 2) OA μg/ml Sample 0.01 0.1 110 100 Ctrl 0.95 9.0 25.7 44.7 54.6 326 0 10.25 23.4 43.9 49.1 327 0 09.1 27.3 40.9 *Values represent percentage contraction compared to thatobtained with 100 μM methacholine (100%), Ctrl = control (0.1% DMSO).

TABLE 7 EFFECTS OF COMPOUND 330 (EACH 30 μM) ON ALLERGEN-INDUCEDCONTRACTIONS OF ISOLATED TRACHEA* (STUDY 3) OA μg/ml Sample 0.001 0.010.1 1 10 100 Ctrl 6.0 12.0 26.0 41.0 54.0 59.0 330 0 1.90 9.00 17.5 26.030.0 *Values represent percentage contraction compared to that obtainedwith 100 μM methacholine (100%), Ctrl = control (0.1% DMSO).

In addition, the effect of compounds of the invention on lung functionin vivo was determined in sensitized animals as follows:

Female Cam Hartley guinea pigs (350–400 g) are sensitized to ovalbuminby exposure of the guinea pigs to a nebulized solution of 1% ovalbuminin saline for 15 minutes. After 10–12 days, the animals are found to beacutely sensitized to the allergen (ovalbumin). Animals are treated byoral gavage, under light halothane anesthesia, with 300 μlpolyethyleneglycol-200 (PEG) or 5 mg/Kg of test compound in 300 μl ofPEG. Animals are treated once daily for 4 days with the final doseadministered 2 hours prior to allergen challenge. Alternatively,compounds were delivered by inhalation, using a Hudson nebulizer drivenby 6 psi oxygen, providing a single dose of 50 μg/Kg 20 min prior tochallenge.

An animal is anaesthetized using ketamine (50 mg/ml; i.p.) and xylazine(10 mg/Kg; i.p.) and 1% halothane during the surgical procedure. Atracheostomy is performed and a water-filled esophageal cannula isinserted prior to positioning the animal in a body plethysmograph. Thetracheal cannula is attached to a fixed tracheal cannula in theplethysmograph. Cardiac function is monitored using electrocardiography.The animal is paralyzed using pancuronium bromide (0.8 mg/Kg; i.m.) andventilated with 3 ml tidal breaths using a Harvard small animalventilator, at a frequency of 60 breaths per minute. Pulmonaryresistance and dynamic lung compliance data are obtained from volume,flow and transpulmonary pressure signals using multipoint analysis.

Pulmonary function is continually monitored throughout the experimentand measurements of lung resistance and lung compliance are made atvarious time-points (e.g., 0, 1, 2, 3, 4, 5, 10, 20 and 30 min)following antigen challenge. Data is collected on a computer-linkedphysiological measurement system using DIREC Physiological recordingsoftware and analyzed using ANADAT software designed for lung mechanicsmeasurements. This software was obtained from RHT-InfoDat Inc.,Montreal, Quebec, Canada.

Once baseline resistance and compliance measurements are obtained, theanimal is challenged with 6 breaths of saline. After 10 minutes, duringwhich time no alterations in lung function should occur, the animal ischallenged with 6 breaths of 2 or 3% ovalbumin in saline (as the antigenstimulus). Saline and antigen are delivered in each breath using aHudson nebulizer. The protective effects of compound 330 administeredorally on OA-induced contraction of tracheal tissue are summarized inTables 8 and 9 below.

TABLE 8 THE EFFECT OF COMPOUND 330 (5 MG/ KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED INCREASE IN LUNG RESISTANCE IN SENSITIZED GUINEA PIGSLung Resistance Time Interval after (cm H₂O/ml/sec) challenge Control330 Baseline 0.287 ± 0.020 0.275 ± 0.036 OA/10 s 0.295 ± 0.024 0.260 ±0.036  1 min 0.982 ± 0.209 0.560 ± 0.101  2 min 2.390 ± 0.728 0.845 ±0.201  3 min 2.627 ± 0.714 0.887 ± 0.160 (P < 0.06)  4 min 2.801 ± 1.0420.778 ± 0.119*  5 min 2.514 ± 0.952 0.791 ± 0.139* 10 min 1.329 ± .2090.661 ± 0.141* 20 min 1.352 ± 0.494 0.366 ± 0.046* 30 min  1.00 ± 0.4340.340 ± 0.037* *Significant difference from control, P < 0.05.

TABLE 9 THE EFFECT OF COMPOUND 330 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED DECREASES IN LUNG COMPLIANCE IN SENSITIZED GUINEA PIGSLung Compliance Time Interval after (ml/cm H₂O) challenge Control 330Baseline 0.412 ± 0.053 0.318 ± 0.042 OA/10 s 0.573 ± 0.083 0.435 ± 0.041 1 min 0.077 ± 0.016 0.182 ± 0.093  2 min 0.029 ± 0.006 0.145 ± 0.092  3min 0.024 ± 0.003 0.133 ± 0.095  4 min 0.023 ± 0.001 0.124 ± 0.088*  5min 0.026 ± 0.002 0.125 ± 0.087* 10 min 0.042 ± 0.002 0.150 ± 0.082* 20min 0.059 ± 0.007 0.184 ± 0.061* 30 min 0.077 ± 0.010 0.196 ± 0.061**Significant difference from control, P < 0.05.

The protective effects of compound 330 administered by inhalation onOA-induced contraction of tracheal tissue are summarized in Tables 10–11below.

TABLE 10 THE EFFECT OF COMPOUND 330 (50 μG/KG; INHALATION) ONALLERGEN-INDUCED INCREASE IN LUNG RESISTANCE IN SENSITIZED GUINEA PIGSLung Resistance Time Interval (cm H₂O/ml/sec) after challenge Control330 Baseline 0.257 ± 0.019 0.300 ± 0.025 OA/10 s 0.257 ± 0.046 0.288 ±0.036  1 min 0.557 ± 0.118 0.382 ± 0.033  2 min 1.323 ± 0.344 0.420 ±0.044*  3 min 1.987 ± 0.572 0.420 ± 0.051*  4 min 1.625 ± 0.248 0.455 ±0.047*  5 min 1.395 ± 0.193 0.446 ± 0.124* 10 min 0.949 ± 0.165 0.436 ±0.036* 20 min 0.589 ± 0.091 0.413 ± 0.076 30 min 0.493 ± 0.067 0.412 ±0.072 *Significant difference from control, P < 0.05.

TABLE 11 THE EFFECT OF COMPOUND 330 (50 μG/KG; INHALATION) ONALLERGEN-INDUCED DECREASE IN LUNG COMPLIANCE IN SENSITIZED GUINEA PIGSLung Compliance Time Interval (ml/cm H₂O) after challenge Control 330Baseline 0.515 ± 0.169 0.463 ± 0.129 OA/10 s 0.526 ± 0.042 0.565 ± 0.062 1 min 0.095 ± 0.015 0.349 ± 0.059*  2 min 0.044 ± 0.010 0.213 ± 0.046* 3 min 0.031 ± 0.007 0.176 ± 0.045*  4 min 0.037 ± 0.009 0.145 ± 0.046* 5 min 0.047 ± 0.007 0.146 ± 0.031* 10 min 0.127 ± 0.053 0.138 ± 0.02220 min 0.096 ± 0.009 0.193 ± 0.054 30 min 0.110 ± 0.010 0.181 ± 0.046*Significant difference from control, P < 0.05.

The protective effects of compound 339 administered orally on OA-inducedcontraction of tracheal tissue are summarized in Tables 12–13 below.

TABLE 12 THE EFFECT OF COMPOUND 339 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED INCREASE IN LUNG RESISTANCE IN SENSITIZED GUINEA PIGSLung Resistance Time Interval (cm H₂O/ml/sec) after challenge Control339 Baseline  0.25 ± 0.008 0.249 ± 0.017 OA/10 s 0.261 ± 0.011 0.239 ±0.013  1 min 1.781 ± 0.737 0.326 ± 0.041*  2 min 3.079 ± 1.066 0.522 ±0.187*  3 min 3.623 ± 0.806 1.102 ± 0.047*  4 min 1.699 ± 0.342 0.996 ±0.380  5 min 2.783 ± 1.010 1.014 ± 0.413 10 min 1.115 ± 0.348 0.440 ±0.099 20 min 0.624 ± 0.178 0.296 ± 0.031 30 min 0.465 ± 0.126 0.291 ±0.037 *Significant difference from control, P < 0.05.

TABLE 13 THE EFFECT OF COMPOUND 339 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED DECREASE IN LUNG COMPLIANCE IN SENSITIZED GUINEA PIGSLung Compliance Time Interval (ml/cm H₂O) after challenge Control 339Baseline 0.548 ± 0.116 0.463 ± 0.026 OA/10 s 0.598 ± 0.129 0.442 ± 0.025 1 min 0.026 ± 0.005 0.172 ± 0.027*  2 min 0.018 ± 0.002 0.088 ± 0.018* 3 min 0.016 ± 0.002 0.060 ± 0.017*  4 min 0.019 ± 0.002 0.050 ± 0.013 5 min 0.021 ± 0.003 0.051 ± 0.011 10 min 0.043 ± 0.005 0.084 ± 0.012*20 min  .074 ± 0.007 0.123 ± 0.015* 30 min 0.093 ± 0.010 0.150 ± 0.012**Significant difference from control, P < 0.05.

The protective effects of compound 342 administered orally on OA-inducedcontraction of tracheal tissue are summarized in Tables 14–15 below.

TABLE 14 THE EFFECT OF COMPOUND 342 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED INCREASE IN LUNG RESISTANCE IN SENSITIZED GUINEA PIGSLung Resistance Time Interval (cm H₂O/ml/sec) after challenge Control342 Baseline 0.214 ± 0.010  0.212 ± 0.020 OA/10 s 0.204 ± 0.010  0.223 ±0.020  1 min 2.380 ± 0.83  0.453 ± 0.120*  2 min 4.241 ± 1.04  1.786 ±0.82*  3 min 4.657 ± 1.21  1.930 ± 0.55*  4 min 4.088 ± 1.42  1.621 ±0.36*  5 min 4.519 ± 1.65 1.4816 ± 0.32* 10 min 1.821 ± 0.38  1.002 ±0.14* 20 min 0.979 ± 0.23  0.524 ± 0.08* 30 min 0.703 ± 0.24  0.354 ±0.04 *Significant difference from control, P < 0.05.

TABLE 15 THE EFFECT OF COMPOUND 342 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ONALLERGEN-INDUCED DECREASE IN LUNG COMPLIANCE IN SENSITIZED GUINEA PIGSLung Compliance Time Interval (ml/cm H₂O) after challenge Control 342Baseline 0.441 ± 0.034 0.444 ± 0.037 OA/10 s 0.509 ± 0.057 0.464 ± 0.031 1 min 0.028 ± 0.007 0.154 ± 0.055*  2 min 0.027 ± 0.012 0.073 ± 0.038* 3 min 0.016 ± 0.004 0.044 ± 0.022*  4 min 0.017 ± 0.004 0.044 ± 0.022 5 min 0.018 ± 0.004 0.038 ± 0.015 10 min 0.034 ± 0.004 0.048 ± 0.005 20min 0.054 ± 0.005 0.084 ± 0.005 30 min 0.074 ± 0.008 0.109 ± 0.006*Significant difference from control, P < 0.05.Effect of Selected Compounds on Allergen Induced Lung Inflammation

The ability of a compound to inhibit the allergen-induced accumulationof inflammatory cells such as eosinophils and neutrophils in the lavagefluid obtained from sensitized animals is indicative of anti-asthmaactivity. In particular, the model system is useful in the evaluation ofthe effects of compounds in the treatment of the late-phase response ofasthma, when lung inflammation and the second phase ofbronchoconstriction is apparent.

Male Brown Norway rats (200–250 g) are sensitized to ovalbumin by anintraperitoneal injection of 1 mg ovalbumin and 100 mg aluminumhydroxide in 1 ml of sterile saline. After 21 days the animals are foundto be sensitized to ovalbumin. Animals are treated with drug or vehicle(0.3 ml PEG-200) once daily for 4 days by oral gavage. The animals arechallenged by exposure, for a period of 60 minutes, to nebulizedsolution of 0.5% ovalbumin in saline generated using a Devillbisnebulizer. The final dose of drug is given 24 hours after challenge.Forty eight hours after challenge the animals are euthanized by anoverdose of halothane and the lungs are lavaged with 7×2 mLs of sterilesaline (room temperature). The recovered lavage fluid is placed on iceand centrifuged at 1200 rpm to separate the cells from the supernatant.The cells are exposed briefly to Tris/ammonium chloride, pH 7.3 toremove any red cells, and washed in phosphate buffered saline. Cytospinsof each cell sample are prepared and stained for the presence of cellscontaining peroxidase and for the determination of the numbers ofeosinophils and neutrophils. The numbers of inflammatory cells areexpressed as a percentage of the total number of cells recovered in thelavage fluid. The protective effect of compound 330 on allergen-inducedlung inflammation is summarized in Table 16.

TABLE 16 EFFECT OF COMPOUND 330 (5 MG/KG/DAY FOR 4 DAYS, P.O.) ONOVALBUMIN-INDUCED ACCUMULATION OF INFLAMMATORY CELLS IN THE LUNG LAVAGEFLUID OBTAINED FROM SENSITIZED BROWN NORWAY RATS# Percentage Total CellsRecovered in Lavage Fluid Cells Stained Positive Treatment forPeroxidase Eosinophils Neutrophils Control  0.55 ± 0.27 0.69 ± 0.300.665 ± 0.31 OA Alone 36.03 ± 5.55 20.0 ± 2.65 11.58 ± 1.53 Cpd 330 + OA 5.65 ± 2.44* 1.98 ± 0.78*  6.16 ± 4.54 #Drug was administered in 300 μLpolyethyleneglycol-200 which was used as a vehicle. No-drug-treatedanimals received 300 μL polyethyleneglycol-200 alone. *Significantdifference from OA alone, P < 0.05.Effect of Selected Compounds in the Allergic Sheep Model of Asthma

Effect of selected compounds in the allergic sheep model of asthma werestudied.

The allergic sheep model was used as it exhibits the cardinal featuresassociated with asthma. Such a model exhibits natural allergy, early(acute) bronchoconstriction, late phase bronchoconstriction, lunginflammation and bronchial hyperresponsiveness. The model is conscious,where the animals are breathing spontaneously, allowing measurement ofairways bronchoconstriction and acute airway hyperresponsiveness.

Sheep which were naturally sensitized to Ascaris suum (30–40 Kg) wereintubated with an endotracheal tube and a balloon catheter, positionedin the lower esophagus. Pleural pressure was estimated with theesophageal catheter, while later pressure was measured with a side-holecatheter advanced through and positioned distal to the tip of theendotracheal tube. Transpulmonary pressure differences between thetrachea and pleural pressures were measured with a differential pressuretransducer catheter system.

The proximal end of the endotracheal tube was connected to a Fleischpneumotachograph in order to measure flow changes. Pulmonary resistance(R_(L)) was calculated from pressure measurements of transpulmonarypressure, respiratory volume (from digital integration of the flowsignal) and flow by the mid-flow technique. SR_(L) was calculated asR_(L)V_(tg) (V_(tg)=thoracic gas volume).

Aerosols generated using a disposable nebulizer, were directed into aT-piece connected to a Harvard respirator and the tracheal tube inseries. The aerosol delivery was controlled using a dosimeter system,consisting of a solenoid valve and compressed air (20 psi) activated atthe start of each inspiratory cycle. Aerosols were delivered in tidalvolumes of 500 ml at 20 Hz.

Selected compounds from the invention were dissolved as a stock solutionin DMSO and diluted in saline. Animals received either, 400 μg/Kg ofcompound 30 min prior to challenge and 4 hours after challenge, or 400μg/Kg of compound for 4 days with the last dose 2 hours beforechallenge. Ascaris suum extract was diluted in phosphate buffered salineto a concentration of 82000 protein nitrogen units/ml and delivered byaerosol over 20 min. Carbachol was dissolved in PBS to concentrations of0.25, 0.5 1.0, 2.0, 4.0% wt/vol. Each animal served as its own controlthroughout the study.

Specific lung resistance (SR_(L)) was measured every 60 min for 8 hoursafter antigen challenge. Airways hyperresponsiveness to carbachol wasmeasured 24 hours after the initial challenge.

The protective effects of compound 330 administered acutely (30 minprior to challenge and 4 hours after challenge; 400 μg/Kg) on specificlung resistance and hyperresponsiveness are summarized in Tables 17–18.

TABLE 17 EFFECT OF COMPOUND 330 (400 μG/KG 30 MIN PRIOR TO CHALLENGE AND4 HRS AFTER CHALLENGE, BY INHALATION) ON ALLERGEN-INDUCED CHANGES INSPECIFIC LUNG RESISTANCE IN ASCARIS SUUM SENSITIZED SHEEP# Specific LungResistance Time Interval after (% Baseline) Challenge (hr) Control 330Baseline  7 ± 10 2 ± 5  −0.5  7 ± 10 0 ± 4 Challenge (0) 266 ± 33 268 ±35  1 197 ± 51 117 ± 11  2  77 ± 21 49 ± 11 3  68 ± 36 32 ± 6  4 23 ± 720 ± 6  5  73 ± 12 14 ± 4* 6 132 ± 29 14 ± 5*   6.5 126 ± 15 18 ± 6* 7129 ± 16 10 ± 2*   7.5 156 ± 13 17 ± 8* 8 123 ± 27  5 ± 3* #Drug wasadministered in 3 mLs (66% DMSO in saline). Vechicle alone had noeffect. Animals were treated once 30 min prior to challenge and 4 hourspost challenge. *Significantly different from control, P < 0.05.

TABLE 18 EFFECT OF COMPOUND 330 (400 μG/KG 30 MIN PRIOR TO CHALLENGE AND4 HOURS AFTER CHALLENGE, BY INHALATION) ON BRONCHIAL HYPERRESPONSIVENESSTO CARBACHOL IN ASCARIS SUUM SENSITIZED SHEEP# PC₄₀₀ (Breath Units)Hyperresponsiveness Control 330 Baseline 26.65 ± 3.08 26.01 ± 3.06Post-Challenge 12.28 ± 1.49 22.79 ± 6.11* #Drug was administered in 3mLs (66% DMSO in saline). Vechicle alone had no effect. Animals weretreated once 30 min prior to challenge and 4 hours post challenge.Hyperresponsiveness to carbachol was measured 24 hours after the initialchallenge. *Significantly different from control, P < 0.05.

Further studies demonstrated the protective effects of compound 330administered for 4 days (400 μg/Kg) on specific lung resistance andhyperresponsiveness are summarized in Tables 19–20.

TABLE 19 EFFECT OF COMPOUND 330 (400 μG/KG/DAY FOR 4 DAYS, BYINHALATION) ON ALLERGEN-INDUCED CHANGES IN SPECIFIC LUNG RESISTANCE INASCARIS SUUM SENSITIZED SHEEP# Specific Lung Resistance Time Intervalafter (% Baseline) Challenge (hr) Control 330 Baseline 2.00 ± 2.00 5.25± 4.50  −0.5 2.00 ± 2.00 −3.75 ± 4.29   Challenge (0) 248.25 ± 85.71 126.00 ± 19.11  1 170.75 ± 58.62  34.00 ± 10.75 2 74.25 ± 17.10 11.50 ±6.18* 3 82.00 ± 6.82   −15.00 ± 37.67*   4 21.25 ± 5.41   4.00 ± 1.78* 557.50 ± 7.51   −4.50 ± 4.17*   6 132.00 ± 9.68   7.75 ± 9.75*   6.5153.75 ± 21.93   5.50 ± 4.87* 7 173.75 ± 21.74  10.75 ± 4.91*   7.5148.00 ± 20.96   4.00 ± 2.35* 8 124.75 ± 28.53   3.25 ± 4.73* #Drug wasadministered in 3 mLs (66% DMSO in saline). Vechicle alone had noeffect. Animals were treated for 4 days with the final dose 30 min priorto challenge. *Significantly different from control, P < 0.05.

TABLE 20 EFFECT OF COMPOUND 330 (400 μG/KG/DAY FOR 4 DAYS, BYINHALATION) ON BRONCHIAL HYPERRESPONSIVENESS TO CARBACHOL IN ASCARISSUUM SENSITIZED SHEEP# PC₄₀₀ (Breath Units) Hyperresponsiveness Control330 Baseline 25.1 ± 1.54 21.26 ± 2.75 Post-Challenge 11.9 ± 1.09 21.21 ±3.10* #Drug was administered in 3 mLs (66% DMSO in saline). Vechiclealone had no effect. Animals were treated for 4 days with the final dose30 min prior to challenge. Hyperresponsiveness to carbachol was measured24 hours after the initial challenge. *Significantly different fromcontrol, P < 0.05.Effects of Selected Compounds on Transcription Factors Involved in theInflammatory Process

The hallmark of a number of chronic inflammatory diseases is theactivation of a number of genes known to be integral in maintaining theinflammation state. Among these are cytokines, chemokines, adhesionmolecules, transcription factors and proteases. Pivotal to the inducedexpression of many of these pro-inflammatory molecules are a class ofproteins called transcription factors. One family of transcriptionfactors known to be key to a pro-inflammatory state is NF-κB. A numberof clinical disease states are associated with elevated levels ofactivated NF-κB. These include atherosclerosis, cancers, infectiousdiseases, and various inflammatory based diseases including asthma,inflammatory bowel disease, arthritis, ischemia/perfusion andinflammatory skin conditions. It was discovered that compounds describedin the invention caused inhibition of NF-κB activation caused by phorbolesters (activators of NF-κB).

Gel shift assays were used to examine the effect of selected compoundsin the invention on the activation of NF-κB, by determining the level ofbinding of NF-κB to specific sites on DNA. Oligonucleotides used tomeasure binding of NF-κB were labeled by the following procedure. 5 μlNF-κB oligonucleotide (8.9 pmol), 2 μl 10×T4-polynucleotide kinasebuffer, 10 units T4 polynucleotide kinase, and 1 ul γ-P-32-dATP (10 μCi)were made up to a final volume of 20 μl with H₂O. The reaction wasincubated at 37° C. for 30 minutes. At this time the reaction wasquenched with 2 μl 0.5 M EDTA and 2 μl 3M NaOAc (pH 5.2). 2.5× vol of100% EtOH was added and the resultant mixture centrifuged at 15,000 g(eppendorf microfuge) for 10 minutes. The pellet was then washed severaltimes in 70% ethanol, air dried at room temperature for 10 minutes, andresuspended in double distilled H₂O (final conc. of 0.75 pmol/2 μl).Cells (RBL-2H3 and A-549) were washed twice in phosphate buffered saline(PBS) at room temperature. They were scraped off the tissue culturedishes into 5 ml PBS using a cell scraper and centrifuged (1500 rpm atroom temp; Beckman GPR centrifuge). Following the removal of thesupernatant the cells were resuspended in 2× pellet volume of Buffer A(0.25M Sucrose, 20 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mMDTT, 0.5 mM Spermidine, 0.15 mM Spermine). This was re-centrifuged andthe cells resuspended in the same buffer at a concentration of 10⁸cells/ml. The cells were allowed to incubate at room temperature for 5minutes. Lysolecithin (10 mg/ml in Buffer A) was added to a finalconcentration of 4001 μg/ml (4 μl/100 μl Buffer A) and the suspensionwas incubated with gentle inversion for no longer than 90 seconds. Celllysis was rapidly stopped by the addition of twice the vol. of ice-coldBuffer A containing 3% BSA. Nuclei were collected by centrifugation at4000 rpm for 1 minute at 4° C. in a microfuge. The supernatant wasremoved and the pellet resuspended in Buffer A containing 3% BSA beforecentrifugation at 30,000 g for 60 seconds at 4° C. (Beckman, TL-100).The nuclei were resuspended in ice-cold Buffer B (20 mM Hepes (pH 7.9),25% v/v glycerol, 0.6 M KCl₂, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.5mM PMSF) at approximately 10⁷ nuclei/ml. The nuclei were disrupted bysonication on ice with 2× five second pulses (40% intensity setting,MICROSON: Ultrasonic cell disrupter). The homogenate was gently stirredon ice for 30 minutes before centrifugation at 25,000 g at 4° C. in aBeckman TL-100. The supernatant was then removed and frozen at −70° C.if not used immediately. Determinations of NF-κB DNA binding activitywere conducted as follows; 2 μl of 10× binding buffer {20 mM HEPES (pH7.5), 50 mM KCl, 5 mM MgCl₂, 200 μg/mg BSA (Sigma # B-2185), 8%glycerol}, 0.4 μl Poly dI-dC (0.5 mg/ml stock), 2.0 μl ³²P-labelledoligo were mixed with 5 μg of protein isolated from cell nuclei. Theresulting mix was made up to a final volume of 20 μl with distilled H₂O.This was then incubated on ice for 5 minutes to allow binding to occur.A further incubation (20 to 30 minutes) at room temperature followed.Samples are then loaded onto a 4.5% acrylamide gel {6 ml (29:1)acrylamide:bis, 2 ml 5×TBE buffer, 800 μl 50% glycerol, 31 ml distilledH₂O, 150 μl 10% APS (ammonium persulphate), 40 μl TEMED}. Acrylamidegels were pre-run in 0.25×TBE buffer for 1.5 hrs (10 volts/cm), followedby a buffer change prior to loading and running of the actual samples.

The effect of selected compounds from the invention on NF-κB activity,as determined using the gel-shift binding assay, are shown in Table 21.

TABLE 21 EFFECT OF SELECTED COMPOUNDS ON NF-κB BINDING IN RBL-2H3 CELLSSTIMULATED WITH TPA (0.1 μM)# Percent Inhibition of Response to TPATreatment (0.1 μM) 165 (10 μM) 54 330 (1 μM) 66 333 (1 μM) 34 339 (1 μM)65 #Compounds or vehicle (0.1% DMSO) were preincubated with cells(RBL-2H3) for 2 hours prior to stimulation with TPA. Cells werestimulated with 0.1 μM TPA for 2.5 hours to activate NF-κB. All valuesshown are as a percent inhibition of control (0.1 μM TPA in the presenceof vehicle).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference.

From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A process for introducing 6α,7β-dioxygenation into a steroid,comprising providing a steroid of Formula (13) having a carbonyl groupat C7 and a double bond between C5 and C6, comprising a reduction thecarbonyl group to a hydroxyl group, followed by a hydroboration of thedouble bond to provide a hydroxyl group at C6, wherein the C6 hydroxylgroup has the α-configuration and the C7 hydroxyl group has theβ-configuration,

wherein each of the compounds of Formulas (13) and (14) includepharmaceutically acceptable salts and solvates thereof, and wherein:each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independentlysubstituted according to any of (a) and (b): (a) one of: ═O, ═C(R⁴)(R⁴),—C(R⁴)(R⁴)(C(R⁴)(R⁴))_(n)— and —(O(C(R⁴)(R⁴))_(n)O)— wherein n rangesfrom 1 to about 6; (b) two of: —X, —R⁴ and —OR¹, each independentlyselected; each of C8, C9, C10, C13 and C14 is independently substitutedwith one of —X, —R⁴ or —OR¹; R¹ is H or a protecting group such that—OR¹ is a protected hydroxyl group, where vicinal —OR¹ groups maytogether form a cyclic structure which protects vicinal hydroxyl groups,and where geminal —OR¹ groups may together form a cyclic structure whichprotects a carbonyl group; R⁴ at each occurrence is independentlyselected from H and C₁₋₃₀ organic moiety that may optionally contain atleast one heteroatom selected from the group consisting of boron,halogen, nitrogen, oxygen, silicon and sulfur, where two geminal R⁴groups may together form a ring with the carbon atom to which they areboth bonded; and X represents fluoride, chloride, bromide and iodide. 2.The process of claim 1 wherein the reduction is accomplished with sodiumborohydride in combination with cerium(III) chloride heptahydrate. 3.The process of claim 1 wherein the hydroboration is conducted with ahydroboration reagent selected from BH₃ and 9-BBN, and in the presenceof an aprotic solvent, followed by treatment with a peroxide selectedfrom hydrogen peroxide and t-butylperoxide, and a base selected fromsodium hydroxide and potassium hydroxide.
 4. The process of claim 3wherein the aprotic solvent is selected from the group consisting oftetrahydrofuran, methylene chloride, diethyl ether, dimethyl sulfide andcarbon disulfide.